Chlorotoxin polypeptides and conjugates and uses thereof

ABSTRACT

Reduced lysine chlorotoxin polypeptides that may be used to generate single species conjugates of chlorotoxin. Conjugates comprising such chlorotoxin polypeptides and pharmaceutical compositions thereof. Methods of using such compositions and/or conjugates.

RELATED APPLICATIONS

This application is a U.S. National Stage Application filed under 35U.S.C. 371, based on International Application No. PCT/US2011/23823,filed Feb. 4, 2011, which claims the benefit of and priority to U.S.Provisional Application No. 61/301,615, filed Feb. 4, 2010, the entirecontents of which are incorporated herein by reference.

BACKGROUND

Chlorotoxin is a peptide component of venom from the scorpion Leiuriusquinquestriatus that has been shown to bind specifically to tumor cells.Chlorotoxin has been used as a targeting agent to deliver cytotoxicand/or imaging agents to a variety of tumors including metastatic tumorsand brain tumors such as malignant glioma. For example, chlorotoxin hasbeen conjugated to the radioactive isotope iodine-131, and suchchlorotoxin conjugates have been shown to be effective anti-tumortherapeutic agents. Other chlorotoxin conjugates including proteinfusions, such as a chlorotoxin-GST fusion protein attached to saporin,have also been shown to result in a significant and selective killing oftumor cells.

SUMMARY

Chlorotoxin can be potentially conjugated to any of a wide variety ofagents, including cytotoxic and/or imaging agents. Although currentlyavailable chlorotoxin conjugates have demonstrated anti-tumorproperties, the present invention encompasses the recognition that agreater repertoire of chlorotoxin conjugates may offer many advantages.To give but one example, different chlorotoxin conjugates may have theirown sets of pharmacokinetic properties that may be particularlydesirable for a given tumor type and/or patient. Furthermore, thepresent invention identifies a previously undocumented source of apotential problem with certain types of chlorotoxin conjugates, stemmingfrom the reality that the wild type chlorotoxin polypeptide (and manyvariants of that polypeptide) contains more than one site at whichconjugation can occur. The present invention therefore provides theinsight that chemical reactions to generate conjugates with chlorotoxinoften result in mixtures of conjugate species. In at least someembodiments, such different species may have different properties.Moreover, efforts to reproduce findings made with such chlorotoxinconjugate mixtures may be hampered by challenges reproducing thedistribution of different species within the preparation. Qualitycontrol, and even analysis, may be difficult or impossible.

The present invention provides new types of chlorotoxin conjugates, andidentifies the source of problems that can be encountered with otherchlorotoxin conjugates. That is, the present invention recognizes thatmany prior chlorotoxin conjugates are prepared by chemically conjugatinga moiety or entity of interest to a chlorotoxin polypeptide. Accordingto the present invention, it is recognized that in at least someinstances, such approaches generate mixed populations of conjugates inwhich moieties or entities of interest are conjugated to chlorotoxin atdifferent points or locations. The present invention encompasses therecognition that such mixed populations may be difficult to characterizeand/or reproduce, and may show different properties (e.g.,pharmacokinetic properties), which differences may be unpredictable. Thepresent invention encompasses the recognition that preparationscontaining only a single species chlorotoxin conjugates may be moredesirable than preparations containing mixtures of conjugates, forexample, in therapeutic and/or diagnostic applications.

The present invention also provides solutions to this identified sourceof a problem. For example, the present invention provides reduced lysinechlorotoxin polypeptides that can generate single species conjugates. Incertain embodiments, provided are reduced lysine chlorotoxinpolypeptides. In various aspects, provided are chlorotoxin conjugatescomprising chlorotoxin polypeptides having not more than one lysineavailable as a site for conjugation (“monolysine chlorotoxinconjugates”), pharmaceutical compositions comprising such conjugates,and methods of using such conjugates. In some embodiments, providedreduced lysine chlorotoxin polypeptides have no lysine residues.

In certain embodiments, the present invention provides methods of makingand of using reduced lysine chlorotoxin polypeptides and conjugatesthereof. In some embodiments, provided reduced lysine chlorotoxinpolypeptides and/or conjugates thereof may be used in medicine (e.g., invarious therapeutic and/or diagnostic contexts).

DEFINITIONS

As used herein, the terms “about” and “approximately,” in reference to anumber, is used herein to include numbers that fall within a range of20%, 10%, 5%, or 1% in either direction (greater than or less than) thenumber unless otherwise stated or otherwise evident from the context(except where such number would exceed 100% of a possible value).

As used herein, the term “characteristic sequence element” or “sequenceelement” refers to a stretch of contiguous amino acids, typically atleast 5 amino acids, e.g., at least 5-50, 5-25, 5-15 or 5-10 aminoacids, that shows at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identity with another polypeptide. In some embodiments, acharacteristic sequence element participates in or confers function on apolypeptide. In some embodiments, reduced lysine chlorotoxinpolypeptides comprise a characteristic sequence element. In some suchembodiments, reduced lysine chlorotoxin polypeptides comprise acharacteristic sequence element that is TTDHQMAR (SEQ ID NO: 29).

The terms “chemotherapeutic,” “anti-cancer agent” and “anti-cancer drug”are used herein interchangeably. They refer to medications that are usedto treat cancer or cancerous conditions. Anti-cancer drugs areconventionally classified in one of the following group: radioisotopes(e.g., Iodine-131, Lutetium-177, Rhenium-188, Yttrium-90), toxins (e.g.,diphtheria, pseudomonas, ricin, gelonin), enzymes, enzymes to activateprodrugs, radio-sensitizing drugs, interfering RNAs, superantigens,anti-angiogenic agents, alkylating agents, purine antagonists,pyrimidine antagonists, plant alkaloids, intercalating antibiotics,aromatase inhibitors, anti-metabolites, mitotic inhibitors, growthfactor inhibitors, cell cycle inhibitors, enzymes, topoisomeraseinhibitors, biological response modifiers, anti-hormones andanti-androgens. Examples of such anti-cancer agents include, but are notlimited to, BCNU, cisplatin, gemcitabine, hydroxyurea, paclitaxel,temozolomide, topotecan, fluorouracil, vincristine, vinblastine,procarbazine, decarbazine, altretamine, methotrexate, mercaptopurine,thioguanine, fludarabine phosphate, cladribine, pentostatin, cytarabine,azacitidine, etoposide, teniposide, irinotecan, docetaxel, doxorubicin,daunorubicin, dactinomycin, idarubicin, plicamycin, mitomycin,bleomycin, tamoxifen, flutamide, leuprolide, goserelin, aminogluthimide,anastrozole, amsacrine, asparaginase, mitoxantrone, mitotane andamifostine.

As used herein, the term “chlorotoxin” refers to a peptide of 36 aminoacids in length, having an amino acid sequence (SEQ ID NO: 1). The term“chlorotoxin” as used herein encompasses chlorotoxin that is isolatedfrom venom of scorpion Leiurius quinquestriatus or other organisms inwhich chlorotoxin may be found, as well as recombinant and syntheticchlorotoxin.

As used herein, the phrase “chlorotoxin conjugate” refers to achlorotoxin polypeptide covalently associated with one or moreentity/entities or moiety/moieties of interest. In some embodiments, theentity of interest is not a polypeptide, and/or is not linked within thepolypeptide chain. In some embodiments, the entity of interest is likedto an amino acid side chain. In some embodiments, the entity of interestis linked to the chlorotoxin polypeptide via a lysine residue.

As used herein, the phrase “chlorotoxin polypeptide” refers to apolypeptide showing at least 45% overall sequence identity withchlorotoxin (SEQ ID NO: 1), and having a length of between twenty fourand forty amino acids, inclusive. In some embodiments, the chlorotoxinpolypeptide has at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% overall sequence identity withSEQ ID NO: 1. In some embodiments, a chlorotoxin polypeptide has atleast 65% overall sequence identity with SEQ ID NO: 1. In someembodiments, a chlorotoxin polypeptide has at least 91% overall sequenceidentity with SEQ ID NO: 1. In some embodiments, a chlorotoxinpolypeptide has at least 94% overall sequence identity with SEQ IDNO: 1. In some embodiments, a chlorotoxin polypeptide has at least 97%overall sequence identity with SEQ ID NO: 1. In some embodiments, achlorotoxin polypeptide further shares at least one characteristicsequence element with SEQ ID NO: 1. In some embodiments, thecharacteristic sequence element is TTDHQMAR (SEQ ID NO: 29). In someembodiments, a chlorotoxin polypeptide has a length between twenty-fourand forty amino acids inclusive. In some embodiments, a “chlorotoxinpolypeptide” includes one or more additional stretch(es) of amino acids,typically at the C- and/or N-terminus and/or as discrete block insertedwithin a sequence. Typically such additional stretches are about 3 toabout 1000 amino acids long. In some embodiments, additional stretchesare about 3-100, 3-90, 3-80, 3-70, 3-60, 3-50, 3-40, 3-30 or 3-20 aminoacids long. In some embodiments, additional stretches are about or lessthan 20 amino acids long, about or less than 15 amino acids long, orabout or less than 10 amino acids long. In some embodiments, theadditional stretch comprises one or more known tags. In someembodiments, the additional stretch comprises a cytotoxic agent.

As used herein, the phrase “combination therapy” refers to theadministration of two or more active agents to the same subject, suchthat the subject is exposed to both agents at the same time. Those ofordinary skill in the art will appreciate that any individual agent maydesirably be administered in a single dose, or in multiple doses, forexample spaced out over predetermined intervals or in a predeterminedpattern. Combination therapy does not require that individual doses ofthe two or more active agents be administered at the same time so longas the subject receiving the doses is simultaneously exposed to bothagents. Combination therapy also does not require that the two or moreactive agents be administered by the same route. In some embodiments,one or both of the at least two active agents administered incombination therapy is administered at a dose and/or frequency that isreduced as compared with its dose or frequency when administered alone.

The phrase “corresponding to,” when used to describe positions or siteswithin amino acid or nucleotide sequences, is used herein as it isunderstood in the art. As is well known in the art, two or more aminoacid or nucleotide sequences can be aligned using standard bioinformatictools, including programs such as BLAST, ClustalX, Sequencher, and etc.Even though the two or more sequences may not match exactly and/or donot have the same length, an alignment of the sequences can still beperformed and, if desirable, a “consensus” sequence generated. Indeed,programs and algorithms used for alignments typically tolerate definablelevels of differences, including insertions, deletions, inversions,polymorphisms, point mutations, etc. Such alignments can aid in thedetermination of which positions in one nucleotide sequence correspondto which positions in other nucleotide sequences.

As used herein, the phrase “dosing regimen” refers to a set of unitdoses (typically more than one) that are administered individuallyseparated by periods of time. The recommended set of doses (i.e.,amounts, timing, route of administration, etc.) for a particularpharmaceutical agent or composition constitutes its dosing regimen.

As used herein, the terms “effective amount” and “effective dose” referto any amount or dose of a compound or composition that is sufficient tofulfill its intended purpose(s), i.e., a desired biological or medicinalresponse in a tissue or subject at an acceptable benefit/risk ratio. Forexample, in certain embodiments of the present invention, the purpose(s)may be: to inhibit angiogenesis, cause regression of neovasculature,interfere with activity of another bioactive molecule, cause regressionof a tumor, inhibit metastases, reduce extent of metastases, etc. Therelevant intended purpose may be objective (i.e., measurable by sometest or marker) or subjective (i.e., subject gives an indication of orfeels an effect). A therapeutically effective amount is commonlyadministered in a dosing regimen that may comprise multiple unit doses.For any particular pharmaceutical agent, a therapeutically effectiveamount (and/or an appropriate unit dose within an effective dosingregimen) may vary, for example, depending on route of administration, oncombination with other pharmaceutical agents. In some embodiments, thespecific therapeutically effective amount (and/or unit dose) for anyparticular patient may depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific pharmaceutical agent employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and/orrate of excretion or metabolism of the specific pharmaceutical agentemployed; the duration of the treatment; and like factors as is wellknown in the medical arts.

As used herein, terms “fluorophore,” “fluorescent moiety,” “fluorescentlabel,” “fluorescent dye” and “fluorescent labeling moiety” are usedherein interchangeably. They refer to a molecule that, in solution andupon excitation with light of appropriate wavelength, emits light back.Numerous fluorescent dyes of a wide variety of structures andcharacteristics are suitable for use in the practice of this invention.Similarly, methods and materials are known for fluorescently labelingnucleic acids (see, for example, R. P. Haugland, “Molecular Probes:Handbook of Fluorescent Probes and Research Chemicals 1992-1994”, 5^(th)Ed., a 1994, Molecular Probes, Inc.). In choosing a fluorophore, it isoften desirable that the fluorescent molecule absorbs light and emitsfluorescence with high efficiency (i.e., high molar absorptioncoefficient and fluorescence quantum yield, respectively) and isphotostable (i.e., it does not undergo significant degradation uponlight excitation within the time necessary to perform the analysis).

As used herein, the term “inhibit” means to prevent something fromhappening, to delay occurrence of something happening, and/or to reducethe extent or likelihood of something happening. Thus, “inhibitingangiogenesis” and “inhibiting the formation of neovasculature” isintended to encompass preventing, delaying, and/or reducing thelikelihood of angiogenesis occiring as well as reducing the number,growth rate, size, etc., of neovessels.

The terms “labeled” and “labeled with a detectable agent or moiety” areused herein interchangeably to specify that an entity (e.g., a reducedlysine chlorotoxin polypeptide or chlorotoxin conjugate) can bevisualized, for example following binding to another entity (e.g., aneoplastic tumor tissue). The detectable agent or moiety may be selectedsuch that it generates a signal which can be measured and whoseintensity is related to (e.g., proportional to) the amount of boundentity. A wide variety of systems for labeling and/or detecting proteinsand peptides are known in the art. Labeled proteins and peptides can beprepared by incorporation of, or conjugation to, a label that isdetectable by spectroscopic, photochemical, biochemical, immunochemical,electrical, optical, chemical or other means. A label or labeling moietymay be directly detectable (i.e., it does not require any furtherreaction or manipulation to be detectable, e.g., a fluorophore isdirectly detectable) or it may be indirectly detectable (i.e., it ismade detectable through reaction or binding with another entity that isdetectable, e.g., a hapten is detectable by immunostaining afterreaction with an appropriate antibody comprising a reporter such as afluorophore). Suitable detectable agents include, but are not limitedto, radionuclides, fluorophores, chemiluminescent agents,microparticles, enzymes, colorimetric labels, magnetic labels, haptens,molecular beacons, aptamer beacons, and the like.

As used herein, the term “macular degeneration” refers to a medicalcondition that results in loss of vision in the center of the visualfield (the macula) because of damage to the retina. Several forms ofmacular degeneration are known to exist, and unless specified, the term“macular degeneration” includes all forms. “Wet macular degeneration”(also known as the neovascular or exudative form) refers to maculardegeneration that involves the growth of blood vessels from the choroidbehind the retina. In wet macular degeneration, the retina may sometimesbecome detached. In “dry macular degeneration” (also known as thenon-exudative form), cellular debris called drusen accumulate betweenthe retina and the choroid, but no blood vessel formation occurs.“Age-related macular degeneration” (ARMD) refers to the most common formof macular degeneration, which typically begins later in life withcharacteristic yellow deposits in the macula. ARMD may occur in eitherthe wet or dry forms of macular degeneration.

As used herein, the term “metastasis” (sometimes abbreviated as “mets”;plural “metastases”) refers to the spread of tumor cells from one organor tissue to another location. The term also refers to tumor tissue thatforms in a new location as a result of metastasis. A “metastatic cancer”is a cancer that spreads from its original, or primary, location, andmay also be referred to as a “secondary cancer” or “secondary tumor.”Generally, metastastic tumors are named for the tissue of the primarytumor from which they originate. Thus, a breast cancer that hasmetastasized to the lung may be referred to as “metastatic breastcancer” even though some cancer cells are located in the lung.

As used herein, the phrase “monolysine chlorotoxin polypeptide” refersto a chlorotoxin polypeptide that has only one lysine residue that isavailable as a site for conjugation. In some embodiments, monolysinechlorotoxin polypeptides have only one lysine residue. In someembodiments, monolysine chlorotoxin polypeptides have more than onelysine residue, but only one of the lysine residues is available as asite for conjugation. In some such embodiments, one or more blockinggroups on some lysines make them unavailable as a site for conjugation.

As used herein, the term “neovasculature” refers to newly formed bloodvessels that have not yet fully matured, i.e., do not have a fullyformed endothelial lining with tight cellular junctions or a completelayer of surrounding smooth muscle cells. As used herein, the term“neovessel” is used to refer to a blood vessel in neovasculature.

The terms “pharmaceutical agent,” “therapeutic agent” and “drug” areused herein interchangeably. They refer to a substance, molecule,compound, agent, factor or composition effective in the treatment,inhibition, and/or detection of a disease, disorder, or clinicalcondition.

A “pharmaceutical composition” is herein defined as a composition thatcomprises an effective amount of at least one active ingredient (e.g., areduced lysine chlorotoxin polypeptide or chlorotoxin conjugate that mayor may not be labeled), and at least one pharmaceutically acceptablecarrier.

As used herein, the term “preventing” when used to refer to the actionof an agent to a process (e.g., angiogenesis, metastasis, cancerprogression, etc.) means reducing extent of and/or delaying onset ofsuch a process when the agent (e.g., a therapeutic agent such as achlorotoxin conjugate) is administered prior to development of one ormore symptoms or attributes associated with the process.

As used herein, the term “primary tumor” refers to a tumor that is atthe original site where the tumor first arose, i.e., as opposed tohaving spread there.

The term “prodrug” refers to a compound that, after in vivoadministration, is metabolized or otherwise converted to thebiologically, pharmaceutically or therapeutically active form of thecompound. A prodrug may be designed to alter the metabolic stability orthe transport characteristics of a compound, to mask side effects ortoxicity, to improve the flavor of a compound and/or to alter othercharacteristics or properties of a compound. By virtue of knowledge ofpharmacodynamic processes and drug metabolisms in vivo, once apharmaceutically active compound is identified, those of skill in thepharmaceutical art generally can design prodrugs of the compound(Nogrady, “Medicinal Chemistry A Biochemical Approach”, 1985, OxfordUniversity Press: N.Y., pages 388-392). Procedures for the selection andpreparation of suitable prodrugs are also known in the art. In someembodiments, a prodrug is a compound whose conversion to its active form(after in vivo administration) involves enzymatic catalysis.

The terms “protein,” “polypeptide” and “peptide” are used hereininterchangeably, and refer to amino acid sequences of a variety oflengths, either in their neutral (uncharged) forms or as salts, andeither unmodified or modified by glycosylation, side chain oxidation, orphosphorylation. In certain embodiments, the amino acid sequence is thefull-length native protein. In other embodiments, the amino acidsequence is a smaller fragment of the full-length protein. In stillother embodiments, the amino acid sequence is modified by additionalsubstituents attached to the amino acid side chains, such as glycosylunits, lipids, or inorganic ions such as phosphates, as well asmodifications relating to chemical conversion of the chains, such asoxidation of sulfhydryl groups. Thus, the term “protein” (or itsequivalent terms) is intended to include the amino acid sequence of thefull-length native protein, subject to those modifications that do notchange its specific properties. In particular, the term “protein”encompasses protein isoforms, i.e., variants that are encoded by thesame gene, but that differ in their pI or MW, or both. Such isoforms candiffer in their amino acid sequence (e.g., as a result of alternativeslicing or limited proteolysis), or in the alternative, may arise fromdifferential post-translational modification (e.g., glycosylation,acylation or phosphorylation).

As used herein, the phrase “reduced lysine chlorotoxin polypeptide”refers to a chlorotoxin polypeptide that has fewer lysine residues thanchlorotoxin (SEQ ID NO: 1) has and/or has fewer lysine residues that areavailable as a site for conjugation than chlorotoxin has. In certainembodiments, a reduced lysine chlorotoxin polypeptide has not more thanone lysine residue. In some embodiments, a reduced lysine chlorotoxinpolypeptide has only one lysine residue. In certain embodiments, areduced lysine chlorotoxin polypeptide has not more than one lysineresidue available as a site for conjugation. In some embodiments, allbut one lysine residue in a reduced lysine chlorotoxin polypeptide havebeen modified such that they are not available as a site forconjugation. In some embodiments, all lysine residues in a reducedlysine chlorotoxin polypeptide have been modified such that they are notavailable as a site for conjugation. In some embodiments, a reducedlysine chlorotoxin polypeptide contains a single site available forconjugation.

The term “regress,” when used to refer to blood vessels and/orvasculature (including neovasculature and/or neovessels), is used hereinto mean to retract, shrink, etc.

The terms “subject” and “individual” are used herein interchangeably.They refer to a human or another mammal (e.g., mouse, rat, rabbit, dog,cat, cattle, swine, sheep, horse or primate) that can be afflicted withor is susceptible to a disease or disorder (e.g., cancer, maculardegeneration, etc.) but may or may not have the disease or disorder. Inmany embodiments, the subject is a human being. In many embodiments, thesubject is a patient. Unless otherwise stated, the terms “individual”and “subject” do not denote a particular age, and thus encompass adults,children, and newborns.

As used herein, the term “susceptible” means having an increased riskfor and/or a propensity for (typically based on genetic predisposition,environmental factors, personal history, or combinations thereof)something, i.e., a disease, disorder, or condition (such as, forexample, cancer, metastatic cancer, macular degeneration, rheumatoidarthritis, etc.) than is observed in the general population. The termtakes into account that an individual “susceptible” for a condition maynever be diagnosed with the condition.

As used herein, the term “systemic administration” refers toadministration of an agent such that the agent becomes widelydistributed in the body in significant amounts and has a biologicaleffect, e.g., its desired effect, in the blood and/or reaches itsdesired site of action via the vascular system. Typical systemic routesof administration include administration by (1) introducing the agentdirectly into the vascular system or (2) oral, pulmonary, orintramuscular administration wherein the agent is adsorbed, enters thevascular system, and is carried to one or more desired site(s) of actionvia the blood.

As used herein, the term “treating” refers to partially or completelyalleviating, ameliorating, relieving, delaying onset of, inhibitingprogression of, reducing severity of, and/or reducing incidence of oneor more symptoms or features of a particular disease, disorder, and/orcondition. For example, “treating” a cancer may refer to inhibitingsurvival, growth, and/or spread of tumor cells; preventing, delaying,and/or reducing the likelihood of occurrence of metastases and/orrecurrences; and/or reducing the number, growth rate, size, etc., ofmetastases. Treatment may be administered to a subject who does notexhibit signs of a disease, disorder, and/or condition and/or to asubject who exhibits only early signs of a disease, disorder, and/orcondition for the purpose of decreasing the risk of developing pathologyassociated with the disease, disorder, and/or condition. In someembodiments, treatment comprises delivery of a pharmaceuticalcomposition to a subject.

As used herein, the phrase “unit dose” refers to a discrete amount of apharmaceutical composition comprising a predetermined amount of anactive ingredient (e.g., a therapeutic agent). The amount of the activeingredient is generally equal to the dosage of the active ingredientthat would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION I. ReducedLysine Chlorotoxin Polypeptides

As shown in Table 1 and as listed in SEQ ID NO: 1, chlorotoxin is a36-amino acid peptide having three lysine residues, at positions 15, 23,and 27 of SEQ ID NO: 1. In certain embodiments, the present inventionprovides chlorotoxin polypeptides having a reduced number of lysineresidues (“reduced lysine chlorotoxin polypeptides”). In certainembodiments, a reduced lysine chlorotoxin polypeptide has an amino acidsequence corresponding to that of SEQ ID NO: 1 in that the reducedlysine chlorotoxin polypeptide has at least 45% overall sequenceidentity with SEQ ID NO: 1 and a length of between twenty-four and fortyamino acid residues inclusive. In some embodiments, a reduced lysinechlorotoxin polypeptide has at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% overall sequenceidentity with SEQ ID NO: 1. In some embodiments, a reduced lysinechlorotoxin polypeptide has at least 65% overall sequence identity withSEQ ID NO: 1. In some embodiments, a chlorotoxin polypeptide has atleast 91% overall sequence identity with SEQ ID NO: 1. For example, areduced lysine chlorotoxin polypeptide may be identical in amino acidsequence to chlorotoxin at 33 out of 36 amino acid residues (i.e.,˜91.7% sequence identity). In some embodiments, a chlorotoxinpolypeptide has at least 94% overall sequence identity with SEQ IDNO: 1. For example, a reduced lysine chlorotoxin polypeptide may beidentical in amino acid sequence to chlorotoxin at 34 out of 36 aminoacid residues (i.e., ˜94.4% sequence identity). In some embodiments, areduced lysine chlorotoxin polypeptide has at least 97% overall sequenceidentity with SEQ ID NO: 1. For example, a reduced lysine chlorotoxinpolypeptide may be identical in amino acid sequence to chlorotoxin at 35out of 36 amino acid residues (i.e., ˜97.2% sequence identity). In someembodiments, a reduced lysine chlorotoxin polypeptide is and/or containsa stretch of 33, 34, 35, 36, 37, or 38 amino acids whose sequencecorresponds to or shows at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%overall sequence identity with the sequence of chlorotoxin.

Table 1 depicts the sequence of chlorotoxin and sequences of exemplaryreduced lysine chlorotoxin polypeptides. Table 1 is not intended to belimiting, but rather, is used to illustrate certain exemplary reducedlysine chlorotoxin polypeptides provided by the present invention.

TABLE 1 Sequences of Chlorotoxin and ofExemplary Reduced Lysine Chlorotoxin Polypeptides SEQ ID NO: CommentSequence (N-terminus to C-terminus) Chlorotoxin 1 Full lengthMCMPC FTTDH QMARK CDDCC GGKGR GKCYG PQCLC R chlorotoxin    5     10    15    20    25    30    35Exemplary reduced lysine polypeptides 2 No lysinesMCMPC FTTDH QMARC DDCCG GGRGC YGPQC LCR    5     10    15    20    25    30 3 No lysinesKMCMP CFTTD HQMAR CDDCC GGGRG CYGPQ CLCR at    5     10    15    20    25    30 positions 15, 23, or 27 of SEQ IDNO: 1; lysine at N-terminus 4 No lysinesMCMPC FTTDH QMARC DDCCG GGRGC YGPQC LCRK at    5     10    15    20    25    30 positions 15, 23 or 27 of SEQ IDNO: 1; lysine at C-terminus 5 Lysines atMCMPC FTTDH QMARA CDDCC GGAGR GACYG PQCLC R positions    5     10    15    20    25    30    35 15, 23, and 27 of SEQ IDNO: 1 replaced by alanine 6 Lysines atMCMPC FTTDH QMARR CDDCC GGRGR GRCYG PQCLC R positions    5     10    15    20    25    30    35 15, 23, and 27 of SEQ IDNO: 1 replaced by arginine 7 Lysines atKMCMP CFTTD HQMAR ACDDC CGGAG RGACY GPQCL CR positions    5     10    15    20    25    30    35 15, 23, and 27 of SEQ IDNO: 1 replaced by alanine; lysine at N-terminus 8 Lysines atKMCMP CFTTD HQMAR RCDDC CGGRG RGRCY GPQCL CR positions    5     10    15    20    25    30    35 15, 23, and 27 of SEQ IDNO: 1 replaced by arginine; lysine at N-terminus 9 Lysines atMCMPC FTTDH QMARA CDDCC GGAGR GACYG PQCLC RK positions    5     10    15    20    25    30    35 15, 23, and 27 of SEQ IDNO: 1 replaced by alanine; lysine at C-terminus 10 Lysines atMCMPC FTTDH QMARR CDDCC GGRGR GRCYG PQCLC RK positions    5     10    15    20    25    30    35 15, 23, and 27 of SEQ IDNO: 1 replaced by arginine; lysine at C-terminus 11 No lysineMCMPC FTTDH QMARC DDCCG GKGRG KCYGP QCLCR at position    5     10    15    20    25    30    35 15 of SEQ ID NO: 1 12No lysine MCMPC FTTDH QMARK CDDCC GGGRG KCYGP QCLCR at position    5     10    15    20    25    30    35 23 of SEQ ID NO: 1 13No lysine MCMPC FTTDH QMARK CDDCC GGKGR GCYGP QCLCR at position    5     10    15    20    25    30    35 27 of SEQ ID NO: 1 14No lysines MCMPC FTTDH QMARC DDCCG GGRGK CYGPQ CLCR at positions    5     10    15    20    25    30 15 and 23 of SEQ ID NO: 1 15No lysines MCMPC FTTDH QMARC DDCCG GKGRG CYGPQ CLCR at positions    5     10    15    20    25    30 15 and 27 of SEQ ID NO: 1 16No lysines MCMPC FTTDH QMARK CDDCC GGGRG CYGPQ CLCR at positions    5     10    15    20    25    30 23 and 27 of SEQ ID NO: 1 17Lysines at MCMPC FTTDH QMARA CDDCC GGAGR GKCYG PQCLC R positions    5     10    15    20    25    30    35 15 and 23 of SEQ ID NO: 1replaced by alanine 18 Lysines atMCMPC FTTDH QMARA CDDCC GGKGR GACYG PQCLC R positions    5     10    15    20    25    30    35 15 and 27 of SEQ ID NO: 1replaced by alanine 19 Lysines atMCMPC FTTDH QMARK CDDCC GGAGR GACYG PQCLC R positions    5     10    15    20    25    30    35 23 and 27 of SEQ ID NO: 1replaced by alanine 20 Lysines atMCMPC FTTDH QMARR CDDCC GGRGR GKCYG PQCLC R positions    5     10    15    20    25    30    35 15 and 23 of SEQ ID NO: 1replaced by arginine 21 Lysines atMCMPC FTTDH QMARR CDDCC GGKGR GRCYG PQCLC R positions    5     10    15    20    25    30    35 15 and 27 of SEQ ID NO: 1replaced by arginine 22 Lysines atMCMPC FTTDH QMARK CDDCC GGRGR GRCYG PQCLC R positions    5     10    15    20    25    30    35 23 and 27 of SEQ ID NO: 1replaced by arginine 23 Lysine atMCMPC FTTDH QMARR CDDCC GGKGR GACYG PQCLC R position    5     10    15    20    25    30    35 15 of SEQ ID NO: 1replaced by arginine; lysine at position 27 of SEQ ID NO: 1 replaced byalanine 24 No lysine MCMPC FTTDH QMARC DDCCG GAGRG ACYGP QCLCRat position     5     10    15    20    25    30    35 15 of SEQ IDNO: 1; lysines at positions 23 and 27 of SEQ ID NO: 1 replaced byarginine 25 No lysine MCMPC FTTDH QMARA CDDCC GGGRG ACYGP QCLCRat position     5     10    15    20    25    30    35 23 of SEQ IDNO: 1; lysines at positions 15 and 27 replaced by arginine 26 No lysineMCMPC FTTDH QMARR CDDCC GGRGR GCYGP QCLCR at position    5     10    15    20    25    30    35 27 of SEQ ID NO: 1;lysines at positions 15 and 23 replaced by arginine

In certain embodiments, reduced lysine chlorotoxin polypeptides have notmore than one lysine available as a site for conjugation. In some suchembodiments, one lysine is available and it is at a position within thechlorotoxin polypeptide that corresponds to a position where a lysine ispresent in chlorotoxin (i.e., position 15, 23 or 27 of SEQ ID NO: 1). Insome embodiments, the single lysine that is available is at position 15of SEQ ID NO: 1. In some embodiments, the single lysine that isavailable is at position 23 of SEQ ID NO: 1. In some embodiments, thesingle lysine that is available is at position 27 of SEQ ID NO: 1. Insome embodiments, a single lysine is present in a reduced lysinechlorotoxin polypeptide of the present invention at a positioncorresponding to a site in chlorotoxin that does not contain a lysineresidue (i.e., not at a position corresponding to any of positions 15,23 or 27 of SEQ ID NO: 1).

In certain embodiments, a reduced lysine chlorotoxin polypeptide lacksat least one amino acid residue corresponding to position 15, 23, or 27of SEQ ID NO: 1.

In certain embodiments, a reduced lysine chlorotoxin polypeptide lackslysine residues entirely. (See, e.g., SEQ ID NOs: 2, 5, 6, 24, 25 and26). In some embodiments, an amino acid is missing where a lysineresidue is normally found in chlorotoxin. In some embodiments, one ormore lysine residues normally found in chlorotoxin is/are replaced byanother amino acid residue and/or by an amino acid derivative. In otherwords, at least one amino acid residue in the polypeptide correspondingto positions 15, 23 or 27 of SEQ ID NO: 1 is not a lysine. In additionto the nineteen other naturally occurring amino acids of whichpolypeptides are typically comprised (alanine, arginine, aspartic acid,asparagine, cysteine, glutamic acid, glutamine, glycine, histidine,isoleucine, leucine, methionine, phenylalanine, proline, serine,threonine, tyrosine, tryptophan, and valine), a variety of substitutesmay be used. Non-limiting examples of substitutes include othernaturally occurring amino acids, non-naturally occurring amino acidssuch as D-amino acids, and amino acid derivatives. Non-limiting examplesof other naturally occurring amino acids include beta-alanine,carnitine, citrulline, cystine, gamma-aminobutyric acid, hydroxyproline,ornithine, and taurine. (For additional examples of naturally occurringamino acids and of amino acid derivatives, see, e.g., Wagner and Musso(1983), “New Naturally Occurring Amino Acids,” Agnew. Chem. Int. Ed.Engl., 22:816-828, the entire of contents of which are hereinincorporated by reference.) In some embodiments, one or more lysineresidues is/are replaced by arginine and/or alanine.

In some embodiments in which a reduced lysine chlorotoxin polypeptidelacks lysine residues entirely, one terminus or both termini (i.e., theN- and/or C-terminus) of the reduced lysine chlorotoxin polypeptide canserve as a site for chemical conjugation. Availability of the terminusor termini may depend on the particular conjugation chemistry employed.In some embodiments in which a reduced lysine chlorotoxin polypeptidelacks lysine residues entirely, only the alpha amino group at theN-terminus is available as a site for conjugation.

In embodiments in which more than one lysine residue is replaced, thesame or different amino acid residue(s) or amino acid derivative(s) maybe used to replace the lysine residues. See, e.g., SEQ ID NOs: 17-22 fornon-limiting examples in which the same amino acid residue has been usedto replace lysine residues and SEQ ID NO: 23 for a non-limiting examplein which different amino acid residues have been used to replace lysineresidues.

In some embodiments, a reduced lysine chlorotoxin polypeptide has onlyone lysine in the sequence (a “monolysine chlorotoxin polypeptide”). Insome embodiments, such a chlorotoxin polypeptide has a lysine residuewhere a lysine residue is normally present in chlorotoxin, e.g., at aposition corresponding to position 15, 23, or 27 of SEQ ID NO: 1 (See,e.g., SEQ ID NOs: 14-23 for non-limiting examples). In some embodiments,a reduced lysine chlorotoxin polypeptide does not have any lysineresidues where a lysine residue is normally present in chlorotoxin(i.e., positions 15, 23, and 27 of SEQ ID NO: 1), but has a lysineresidue at a position that does not correspond to any of positions 15,23, and 27 of SEQ ID NO: 1. As with chlorotoxin polypeptides having nolysines at all, monolysine chlorotoxin polypeptides may be missing aminoacids at one or more positions corresponding to positions 15, 23 and 27of SEQ ID NO: 1, and/or may have an amino acid or amino acid derivativesubstition at one or more positions corresponding to positions 15, 23and 27 of SEQ ID NO: 1.

In some embodiments, provided reduced lysine chlorotoxin polypeptideshave an amino acid sequence that includes one or more than one lysineresidues but nonetheless have a reduced number of lysines available forconjugation when compared with chlorotoxin. In some embodiments, one ormore lysine residues in a reduced lysine chlorotoxin polypeptideprovided herein is/are made unavailable as a site for conjugation thoughthey are present in the chlorotoxin polypeptide. For example, one ormore lysine residue(s) can be covalently or non-covalently modified suchthat the one or more lysine residue(s) is/are blocked from participatingin a chemical conjugation reaction, leaving fewer than 3, 2 or 1 (i.e.,“reduced” lysine) lysine residue(s) available as a site for conjugation.Non-limiting examples of covalent modifications to lysine residues thatcould be employed in this manner include pegylation (i.e., modificationby attachment of a polyethylene glycol polymer), methylation (includingdi- and tri-methylation), and attachment of other alkyl group(s). Incertain embodiments, one or more lysine residues is/are modified at theepsilon NH₂ group. For example, if a given R group (e.g., butyl, propyl,or ethyl group) is used to covalently modify a lysine residue, theepsilon NH₂ group can be modified to an NR₂ or NR₃ group.

Table 2 presents some non-limiting examples of modification schemes thatcould be used to produce reduced lysine chlorotoxin polypeptides.

TABLE 2 Exemplary modification schemes Position(s) of SEQ ID lysine NO:Core sequence (N-terminus to C-terminus) residue(s)  1MCMPC FTTDH QMARK CDDCC GGKGR GKCYG PQCLC R 15, 23 and 27    5     10    15    20    25    30    35 15 and 23 15 and 27 23 and 2711 MCMPC FTTDH QMARC DDCCG GKGRG KCYGP QCLCR 22 and 26    5     10    15    20    25    30    35 22 26 12MCMPC FTTDH QMARK CDDCC GGGRG KCYGP QCLCR 15 and 26    5     10    15    20    25    30    35 15 26 13MCMPC FTTDH QMARK CDDCC GGKGR GCYGP QCLCR 15 and 23    5     10    15    20    25    30    35 15 23 27KMCMP CFTTD HQMAR KCDDC CGGKG RGKCY GPQCL CR 16, 24 and 28 (lysine    5     10    15    20    25    30    35 added to N-term) 28MCMPC FTTDH QMARK CDDCC GGKGR GKCYG PQCLC RK 15, 23 and 27 (lysine    5     10    15    20    25    30    35 added to C-term)

In certain embodiments, blocking of particular lysine residue isachieved by incorporating a modified lysine (in which sites availablefor conjugation are already blocked) during the appropriate step duringsynthesis of the reduced lysine chlorotoxin polypeptide. Modifiedlysines are readily available commercially and can be synthesized byroutine methods known in the art. Non-limiting examples of modifiedlysines that can be used in this manner include, but are not limited to,di-substituted lysine or trisubstitited lysines (e.g., N,N—R₂-lysine orN,N,N—R₃-lysine, where R is the blocking group) and lysines with shortPEG molecules attached to them. R can be any group that when covalentlyattached to the lysine would serve to block the lysine residue fromparticipating in a chemical conjugation reaction. For example, alkylgroups (e.g., butyl, methyl, and ethyl) may serve as blocking groups.For example, N,N-dimethyl-lysine and/or N,N,N-trimethyl-lysine be usedduring synthesis.

In certain embodiments, one terminus or both termini (i.e., the N-and/or C-terminus) of the reduced lysine chlorotoxin polypeptide isblocked so as to prevent the terminus/termini from participating in achemical conjugation reaction. For example, in some embodiments, aconjugation chemistry is used in which at least one terminus wouldparticipate in the conjugation reaction if it were not blocked. Avariety of methods of blocking N- and/or C-termini of polypeptides areknown in the art, including, but not limited to, covalent modificationby the addition of alkyl groups (e.g., methylation) at amines.

Methods of synthesizing reduced lysine chlorotoxin polypeptides asdescribed herein are known in the art. In some peptide synthesismethods, an amino group of one amino acid (or amino acid derivative) islinked to a carboxyl group of another amino acid (or amino acidderivative) that has been activated by reacting it with a reagent suchas dicyclohexylcarbodiimide (DCC). When the free amino group attacks theactivated carboxyl group, a peptide bond is formed and dicyclohexylureais released. In such methods, other potentially reactive groups (such asthe α-amino group of the N-terminal amino acid or amino acid derivativeand the carboxyl group of the C-terminal amino acid or amino acidderivative) may be blocked (“protected”) from participating in thechemical reaction. Thus, only particular active groups react such thatthe desired product is formed. Blocking groups useful for this purposeinclude without limitation tertbutoxycarbonyl groups (t-Boc) andbenzoyloxycarbonyl groups to protect amine groups; and simple esters(such as methyl and ethyl groups) and benzyl esters to protect carboxylgroups. Blocking groups can typically be subsequently removed with atreatment that leaves peptide bonds intact (for example, treatment withdilute acid). This process of protecting reacting groups that should notreact, coupling to form a peptide bond, and deprotecting reactive groupsmay be repeated. A peptide may be synthesized by sequentially addingamino acids to a growing peptide chain. Both liquid-phase and solidphasepeptide synthesis methods are suitable for use in accordance with theinvention. In solid-phase peptide synthesis methods, the growing peptidechain is typically linked to an insoluble matrix (such as, for example,polystyrene beads) by linking the carboxyterminal amino acid to thematrix. At the end of synthesis, the peptide can be released from thematrix using a cleaving reagent that does not disrupt peptide bonds,such as hydrofluoric acid (HF). Protecting groups are also typicallyremoved at this time. Automated, high throughput, and/or parallelpeptide synthesis methods may also be used in accordance with theinvention. For more information about peptide synthesis methods, see,e.g., Merrifield (1969) “Solid-phase peptide synthesis,” Adv EnzymolRelat Areas Mol. Biol., 32:221-96; Fridkin et al. (1974) Annu RevBiochem., 43 (O):419-43; Merrifield (1997) “Concept and EarlyDevelopment of Solid Phase Peptide Synthesis,” Methods in Enzymology,289:3-13; Sabatino et al. (2009) “Advances in automatic, manual andmicrowave-assisted solid-phase peptide synthesis,” Curr Opin Drug DiscovDevel., 11(6):762-70, the entire contents of each of which are hereinincorporated by reference.

In some embodiments, modifications to lysine residues are used incombination with other means as described herein (e.g., replacement of alysine residue with another amino acid or amino acid derivative and/orlack of a lysine residue where one is normally found in chlorotoxin).

In some embodiments, the protecting group in the N-terminus is notremoved at the end of synthesis. Leaving the protecting group on may,for example, serve to generate a reduced lysine chlorotoxin polypeptidewith a blocked N-terminus, thus limiting the sites available forconjugation in a particular chemical conjugation scheme.

II. Chlorotoxin Conjugates

In certain embodiments, provided are chlorotoxin conjugates comprising areduced lysine chlorotoxin polypeptide associated with one or moreentities or moieties. Chlorotoxin conjugates of the present inventioncan include a polypeptide of any length that comprises a reduced lysinechlorotoxin polypeptide as described herein.

A. Conjugation

In some embodiments, the one or more entities or moieties is/areassociated with reduced lysine chlorotoxin polypeptides via a lysineresidue and/or via a terminus of the reduced lysine chlorotoxinpolypeptide. In some such embodiments, the position(s) where entities ormoieties can be attached to a reduced lysine chlorotoxin polypeptide islimited by the number of lysine residues available as a site forconjugation. For example, entities or moieties can be attached at thesingle available lysine residue in monolysine chlorotoxin polypeptides.

In some embodiments, entities or moieties are associated at theN-terminus of or at the C-terminus of the reduced lysine chlorotoxinpolypeptide. In some such embodiments, the reduced lysine chlorotoxinpolypeptide does not have any lysine residues available for conjugationat any of the “native” positions within chlorotoxin (e.g., positionscorresponding to positions 15, 23 and 27).

In some embodiments, the reduced lysine chlorotoxin polypeptide iscovalently associated to the one or more entity/entities ormoeity/moieties. As will be appreciated by those skilled in the art, areduced lysine chlorotoxin polypeptide and one or more entity/entitiesand/or moiety/moieties may be attached either directly or indirectly(e.g., through a linker).

A variety of conjugation chemistries are known in the art and may beused in the practice of the present invention. In certain embodiments,one or more entity/entities or moiety/moieties are attached to theepsilon amino group of a lysine residue. In some embodiments, theconjugation chemistry is based on NHS (N-hydroxysuccinimide)/EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) chemistry.In some embodiments, the conjugation chemistry is based on thiolationchemistry, i.e., using a thiolating agent such as Traut's reagent and/or2-Iminothiolane.

In certain embodiments, a reduced lysine chlorotoxin polypeptide and oneor more entity/entities or moiety/moieties are directly, covalently,linked to each other. Such direct covalent binding can be achieved inany of a variety of ways, for example, via amide, ester, carbon-carbon,disulfide, carbamate, ether, thioether, urea, amine, or carbonate bonds.Such covalent binding can be achieved, for example, by taking advantageof function groups present on the entity/entities or moiety/moietiesand/or on the reduced lysine chlorotoxin polypeptide. Suitablefunctional groups that can be used include, but are not limited to,amines, anhydrides, hydroxyl groups, carboxyl groups, thiols, and thelike. In certain embodiments, a functional group of one part of thefuture conjugate is activated for coupling to the other part of thefuture conjugate. For example, an activating agent, such as acarbodiimide, can be used to effect such a coupling. A wide variety ofactivating agents are known in the art and are suitable for forming aprovided conjugate.

In some embodiments, a reduced lysine chlorotoxin polypeptide and one ormore entity/entities or moiety/moieties are indirectly covalently linkedto each other via a linker group. Such indirect covalent linkage can beaccomplished by using any of a number of stable bifunctional agents wellknown in the art, including homofunctional and heterofunctional agents.For non-limiting examples of such agents, see, e.g., Pierce Catalog andHandbook. Use of a bifunctional agent differs from use of an activatingagent in that the former results in a linking moiety being present inthe resulting conjugate, whereas the latter results in a direct couplingbetween two moieties involved in the reaction. A role of thebifunctional agent may be to allow a reaction between two otherwiseinert moieties. Alternatively or additionally, the bifunctional agent,which becomes part of the reaction product, may be selected such that inconfers some degree of conformational flexibility to the conjugate. Forexample, the bifunctional agent may comprise a straight alkyl chaincontaining several atoms, for example, between 2 and 10 carbon atoms.Alternatively or additionally, the bifunctional agent may be selectedsuch that the linkage formed between the reduced lysine chlorotoxinpolypeptide and the one or more entity/entities or moiety/moieties iscleavable, e.g., hydrolysable. (For non-limiting examples of suchlinkers, see, e.g., U.S. Pat. Nos. 5,773,001; 5,739,116 and 5,877,296,the contents of each of which is incorporated herein by reference.) Suchlinkers may, for example, be used when the entity or moiety beingconjugated to the reduced lysine chlorotoxin polypeptide is atherapeutic moiety that is observed to have a higher activity afterhydrolysis from the reduced lysine chlorotoxin polypeptide. Exemplarymechanisms to achieve cleavage include hydrolysis in the acidic pH oflysosomes (hydrazones, acetals, and cis-aconitate-like amides), peptidecleavage by lysosomal enzymes (e.g., cathepsins and other lysosomalenzymes), and reduction of disulfides. Additional cleavage mechanismsinclude hydrolysis at physiological pH extra- or intracellularly. Thismechanism may be applied when the crosslinker used to couple the one ormore entity/entities or moiety/moieties to the reduced lysinechlorotoxin polypeptide is a biodegradable/bioerodible entity, such aspolydextran and the like.

For example, for conjugates comprising one or more therapeutic moieties,hydrazone-containing conjugates can be made with introduced carbonylgroups that provide the desired drug-release properties. Conjugates canalso be made with a linker that comprises an alkul chain with adisulfide group at one end and a hydrazine derivative at the other end.

Linkers containing functional groups other than hydrazones also have thepotential to be cleaved in the acidic miliey of lysosomes. For example,conjugates can be made from thiol-reactive linkers that contain a groupother than a hydrazone that is cleavable intracellularly, such asesters, amides, and acetals/ketals. Ketals made from a 5 to 7 memberring ketone that has one of the oxygen atoms attached to the entity ormoiety and the other to a linker for attachment to a reduced lysinechlorotoxin polypeptide can also be used.

A further example of class pH-sensitive linkers are the cis-aconitates,which have a carboxylic acid group juxtaposed to an amide group. Thecarboxylic acid accelerates amide hydrolysis in the acidid lysosomes.Linkers that achieve a similar type of hydrolysis rate acceleration withseveral other types of structures can also be used.

Enzymatic hydrolysis of peptides by lysosomal enzymes may also be usedto release entities or moieties from conjugates. For example, a reducedlysine chlorotoxin polypeptide can be attached via an amide bond topara-aminobenzyl alcohol and then a carbamate or carbonate can be madebetween the benzyl alcohol and the entity or moiety. Cleavage of thereduced lysine chlorotoxin polypeptide leads to collapse of the aminobenzyle carabamate or carbonate, and release of the entity or moeity. Asa further example, a phenol can be cleaved by collapse of the linkerinstead of the carbamate. As a yet further example, disulfide reactionis used to initiate collapse of a para-meraptobenzyl carbamate orcarbonate.

Many therapeutic moieties, in particular anti-cancer agents, havelittle, if any, solubility in water, which limits drug loading on aconjugate due to aggregation of the therapeutic moiety. One approach toovercoming this is to add solubilizing groups to the linker Conjugatesmade with a linker consisting of PEG (polyethylene glycol) and adipeptide can be used, including, for example, those having a PEGdi-acid thiol-acid, or maleimide-acid attached to the reduced lysinechlorotoxin conjugate, a dipeptide spacer, and an amide bound to theentity or moiety. Approaches that incorporate PEG groups may bebeneficial in overcoming aggregation and limits in drug loading.

In embodiments in which entity or moiety within a chlorotoxin conjugateis a protein, polypeptide, or peptide, the chlorotoxin conjugate may bea fusion protein. A fusion protein is a molecule comprising two or moreproteins, polypeptides, or peptides linked by a covalent bond via theirindividual peptide backbones. Fusion proteins used in methods of thepresent invention can be produced by any suitable method known in theart. For example, they can be produced by direct protein syntheticmethods using a polypeptide synthesizer. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments that can subsequently be annealed and re-amplified to generatea chimeric gene sequence. Fusion proteins can be obtained by standardrecombinant methods (See, for example, Maniatis et al. “MolecularCloning: A Laboratory Manual,” 2^(nd) Ed., 1989, Cold Spring HarborLaboratory, Cold Spring, N.Y., the entire contents of which are hereinincorporated by reference). These methods generally comprise (1)construction of a nucleic acid molecule that encodes the desired fusionprotein; (2) insertion of the nucleic acid molecule into a recombinantexpression vector; (3) transformation of a suitable host cell with theexpression vector; and (4) expression of the fusion protein in the hostcell. Fusion proteins produced by such methods may be recovered andisolated, either directly from the culture medium or by lysis of thecells, as known in the art. Many methods for purifying proteins producedby transformed host cells are well-known in the art. These include, butare not limited to, precipitation, centrifugation, gel filtration, andcolumn chromatography (e.g., ionexchange, reverse-phase, and affinity).Other purification methods have been described (See, for example,Deutscher et al. “Guide to Protein Purification” in Methods inEnzymology, 1990, vol. 192, Academic Press, the entire contents of whichare herein incorporated by reference).

In certain embodiments, the reduced lysine chlorotoxin polypeptide isnoncovalently associated to the one or more entity/entities ormoiety/moieties. Examples of non-covalent associations include, but arenot limited to, hydrophobic interactions, electrostatic interactions,dipole interactions, van der Waals interactions, and hydrogen bonding.

Irrespective of the nature of the association between the toxin moietyand therapeutic agent, the association is typically selective, specific,and strong enough so that the conjugate does not dissociate before orduring transport to and into cells. Association between a reduced lysinechlorotoxin polypeptide and one or more entity/entities ormoiety/moieties may be achieved using any chemical, biochemical,enzymatic, or genetic coupling known to one skilled in the art.

As can readily be appreciated by those skilled in the art, a conjugateof the present invention can comprise any number of reduced lysinechlorotoxin polypeptides and any number of entities or moeities,associated to one another by any number of different ways. The design ofa conjugate will be influenced by its intended purpose(s) and theproperties that are desirable in the particular context of its use.Selection of a method to associate or bind a reduced lysine chlorotoxinpolypeptide to an entity or moiety to form a conjugate is within theknowledge of one skilled in the art and will generally depend on thenature of the association desired (i.e., covalent vs. non-covalentand/or cleavable vs. non-cleavable), the nature of the reduced lysinechlorotoxin polypeptide and the entity/moiety, the presence and natureof functional chemical groups, and the like.

B. Entities/Moieties

As mentioned above, in certain embodiments, chlorotoxin conjugatescomprise one or more non-chlorotoxin entities. Any of a variety of suchentities or moieties may be employed.

1. Therapeutic Entities/Moieties

In certain embodiments, provided conjugates comprise one or moretherapeutic entities or moeities, as described below and inInternational Patent Publication No. WO 2009/021136 A1, the entirecontents of which are herein incorporated by reference in theirentirety.

a. Anti-Cancer Agents

In some embodiments, the one or more therapeutic entities or moietiescomprises an anti-cancer agent. Suitable anti-cancer agents include anyof a large variety of substances, molecules, compounds, agents orfactors that are directly or indirectly toxic or detrimental to cancercells, including, for example, cytotoxic agents. Anti-cancer agentssuitable for use in the practice of the invention may be synthetic ornatural. Anticancer agents may comprise a single molecule or a complexof different molecules.

Suitable anti-cancer agents can belong to any of various classes ofcompounds including, but not limited to, small molecules, peptides,saccharides, steroids, antibodies, fusion proteins, antisensepolynucleotides, ribozymes, small interfering RNAs, peptidomimetics, andthe like. Similarly, suitable anti-cancer agents can be found among anyof a variety of classes of anti-cancer agents including, but not limitedto, alkylating agents, anti-metabolite drugs, anti-mitotic antibiotics,alkaloidal anti-tumor agents, hormones and anti-hormones, interferons,non-steroidal anti-inflammatory drugs, and various other anti-tumoragents.

Particularly suitable anti-cancer agents are agents that causeundesirable side effects due to poor selectivity/specificity for cancercells; agents that undergo no or poor cellular uptake and/or retention;agents that are associated with cellular drug resistance; and agentsthat cannot be readily formulated for administration to cancer patientsdue to poor water solubility, aggregation, and the like.

Examples of suitable anti-cancer agents that can be used in conjugatesof the present invention are described in more detail below.

Poorly Water Soluble Anti-Cancer Drugs

In certain embodiments, an anti-cancer agent within an inventiveconjugate is a poorly water soluble compound. As will be recognized byone skilled in the art, a wide variety of poorly water solubleanti-cancer agents are suitable for use in the present invention.

For example, an anti-cancer agent may be selected among taxanes, whichare recognized as effective agents in the treatment of many solid tumorsthat are refractory to other anti-neoplastic agents. Two currentlyapproved taxanes are paclitaxel (TAXOL™) and docetaxel (TAXOTERE™).Paclitaxel, docetaxel, and other taxanes act by enhancing thepolymerization of tubulin, an essential protein in the formation ofspindle microtubules. Polymerization of tubulin results in the formationof very stable, nonfunctional tubules, which inhibits cell replicationand leads to cell death.

Paclitaxel is very poorly water soluble, and therefore, cannot bepractically formulated with water for intravenous administration. Someformulations of TAXOL™ for injection or intravenous infusion have beendeveloped using CREMOPHOR EL™ (polyoxyethylated castor oil) as a drugcarrier. However, CREMOPHOR™ EL is itself toxic, and is considered tobe, at least in part, responsible for the hypersensitivity reactions(severe skin rashes, hives, flushing, dyspnea, tacchycardia and others)associated with administration of such preparations. To avoid such sideeffects, premedication is often prescribed along with paclitaxelformulations containing CREMOPHOR™. Docetaxel, which is an analog ofpaclitaxel, is like paclitaxel poorly soluble in water. The currentlymost preferred solvent used to dissolve docetaxel for pharmaceutical useis polysorbate 80 (TWEEN® 80). In addition to causing hypersensitivityreactions in patients, TWEEN® 80 cannot be used with PVC deliveryapparatus, because of its tendency to leach diethylhexyl phthalate,which is highly toxic.

A conjugate according to the present invention comprising a taxane andchlorotoxin polypeptide can be used as an improved delivery method toavoids the use of solvents and carriers that induce adverse reactions inpatients.

In some embodiments, an anti-cancer agent within a chlorotoxin conjugatemay belong to the enediyne family of antibiotics. As a family, theenediyne antibiotics are particularly potent anti-tumor agents. Somemembers are 1000 times more potent than adriamycin, one of the mosteffective, clinically used anti-tumor antibiotics (Y. S. Zhen et al., J.Antibiot., 1989, 42: 1294-1298). For example, an anti-cancer agentwithin an inventive conjugate may be a member of the enediyne family ofcalicheamicins. Originally isolated from a broth extract of the soilmicroorganism Micromonospora echinospora ssp. calichensis, thecalicheamicins were detected in a screen for potent DNA damaging agents(M. D. Lee et al., J. Am. Chem. Soc., 1987, 109: 3464-3466; M. D. Lee etal., J. Am. Chem. Soc., 1987, 109: 3466-3468; W. M. Maiese et al., J.Antibiot., 1989, 42: 558-563; M. D. Lee et al., J. Antibiot., 1989, 42:1070-1087).

Calicheamicins are characterized by a complex, rigid bicyclic enediyneallylic trisulfide core structure linked through glycosyl bonds to anoligosaccharide chain. The oligosaccharide portion contains a number ofsubstituted sugar derivatives, and a substituted tetrahydropyran ring.The enediyne containing core (or aglycone) and carbohydrate portions ofcalicheamicins have been reported to carry out different roles in thebiological activity of these molecules. It is generally believed thatthe core portion cleaves DNA, whereas the oligosaccharide portion of thecalicheamicins serves as a recognition and delivery system and guidesthe drug to a double-stranded DNA minor groove in which the drug anchorsitself (“Enediyne Antibiotics as Antitumor Agents”, Doyle and Borders,1995, Marcel-Dekker: New York). Double-stranded DNA cleavage is a typeof damage that is usually non-repairable or non-easily repairable forthe cell and is most often lethal.

Because of their chemical and biological properties, several analoguesof the calicheamicins have been tested in preclinical models aspotential anti-tumor agents. Their development as single agent therapieshas not been pursued because of delayed toxicities that limit thetherapeutic dose range for treatment. However, their potency makes themparticularly useful for targeted chemotherapy.

Other examples of suitable poorly water soluble anti-cancer agentsinclude tamoxifen and BCNU. Tamoxifen has been used with varying degreesof success to treat a variety of estrogen receptor positive carcinomassuch as breast cancer, endometrial carcinoma, prostate carcinoma,ovarian carcinoma, renal carcinoma, melanoma, colorectal tumors, desmoidtumors, pancreatic carcinoma, and pituitary tumors. In addition to beinglimited by poor water solubility, chemotherapy using tamoxifen can causeside effects such as cellular drug resistance. BCNU(1,3-bis(2-chloroethyl)-1-nitrosourea) is well known for its anti-tumorproperties and, since 1972, it has been charted by the National CancerInstitute for use against brain tumors, colon cancer, Hodgkin's Disease,lung cancer and multiple myeloma. However, the efficient use of thisanti-cancer drug is also compromised by its low solubility.

Anti-Cancer Agents Associated with Drug Resistance

In certain embodiments of the present invention, chlorotoxin conjugatescomprise an anti-cancer agent associated with drug resistance. As usedherein, the term “anti-cancer agent associated with drug resistance”refers to any chemotherapeutic to which cancer cells are or can becomeresistant. As already mentioned above, resistance to an anti-canceragent can be due to many factors and can operate by differentmechanisms. Administration of a conjugate of the present inventioncomprising a reduced lysine chlorotoxin polypeptide and an anti-canceragent associated with drug resistance can enhance cellular uptake of theanti-cancer agent and carry it into tumor cells, e.g., resistant tumorcells.

Any of a wide variety of anti-cancer agents associated with drugresistance are suitable for use in the present invention. For example,the anti-cancer agent associated with drug resistance may bemethotrexate. Methotrexate, a widely used cancer drug, is an analogue offolic acid and blocks important steps in the synthesis oftetrahydrofolic acid which itself is a critical source of compoundsutilized in the synthesis of thymidylate, a building block that isspecific and therefore especially critical for DNA synthesis.Methotrexate-induced drug resistance is linked to a deficiency incellular uptake of that drug.

Other examples of suitable anti-cancer agents include purine andpyrimidine analogs that are associated with drug resistance due toinadequate intracellular activation of the drug through loss ofenzymatic activity. An example of such a purine analog is6-mercaptopurine (6-MP). A common cause of tumor cell resistance to 6-MPis the loss of the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT) which activates 6-MP into its correspondingnucleotide, 6-mercaptophosphoribosylpurine (6-MPRP), the lethal form ofthe drug. Without being held to theory, it is postulated that resistancecould be overcome if 6-MPRP itself could be introduced into the cell.Although this compound is commercially available, it has not yet beenused therapeutically in cancer treatment because it is not adequatelytransported into living cells. Association of 6-MPRP to a reduced lysinechlorotoxin polypeptide according to the present invention woulddramatically increase its ability to cross the cell membrane.Thioguanine is another example of anti-cancer agent that is associatedwith drug resistance due to lack of the enzyme HGPRT.

Examples of pyrimidine analogs that are associated with drug resistancedue to inadequate intracellular activation include cytosine arabinosideand adenosine arabinoside which are activated by the enzymedeoxycytidine kinase (DOCK) to the lethal forms cytosine diphosphate andadenosine diphosphate, respectively. A reduced lysine chlorotoxinpolypeptide can be coupled to the activated form of such pyrimidineanalogs to enhance their cellular uptake and overcome cellular drugresistance.

Other examples of anti-cancer agents associated with drug resistanceinclude, but are not limited to, 5-fluorouracil, fluorodeoxyuridine,cytosine, arabinoside, vinblastin, vincristin, daunorubicin,doxorubicin, actinomycin, and bleomycin.

Other Anti-Cancer Agents

In some embodiments, an anti-cancer agent is selected from the groupconsisting of alkylating drugs (e.g., mechlorethamine, chlorambucil,cyclophosphamide, melphalan, ifosfamide), antimetabolites (e.g.,methotrexate), purine antagonists and pyrimidine antagonists (e.g.,6-mercaptopurine, 5-fluorouracil, cytarabile, gemcitabine), spindlepoisons (e.g., vinblastine, vincristine, vinorelbine, paclitaxel),podophyllotoxins (e.g., etoposide, irinotecan, topotecan), antibiotics(e.g., doxorubicin, bleomycin, mitomycin), nitrosoureas (e.g.,carmustine, lomustine), inorganic ions (e.g., cisplatin, carboplatin),enzymes (e.g., asparaginase), and hormones (e.g., tamoxifen, leuprolide,flutamide, and megestrol), to name a few. For a more comprehensivediscussion of updated cancer therapies see the website whose address is<http://www.cancer.gov/>, a list of the FDA approved oncology drugs atthe website whose address is<http://www.fda.gov/cder/cancer/druglistframe.htm>, and The MerckManual, Seventeenth Ed. 1999, the entire contents of which are herebyincorporated by reference.

Nucleic Acid Agents

In certain embodiments, chlorotoxin conjugates comprise a nucleic acidagent.

Numerous cancers and tumors have been shown to be associated withvarying degrees of genetic impairment, such as point mutations, genedeletions, or duplications. Many new strategies for the treatment ofcancer, such as those that have been termed “antisense,” “antigene” and“RNA interference” have been developed to modulate the expression ofgenes (A. Kalota et al., Cancer Biol. Ther., 2004, 3: 4-12; Y. Nakata etal., Crit. Rev. Eukaryot. Gene Expr., 2005, 15: 163-182; V. Wacheck andU. Zangmeister-Wittke, Crit. Rev. Oncol. Hematol., 2006, 59: 65-73; A.Kolata et al., Handb. Exp. Pharmacol., 2006, 173: 173-196). Theseapproaches utilize, for example, antisense nucleic acids, ribozymes,triplex agents, or short interfering RNAs (siRNAs) to block thetranscription or translation of a specific mRNA or DNA of a target gene,either by masking that mRNA with an antisense nucleic acid or DNA with atriplex agent, by cleaving the nucleotide sequence with a ribozyme, orby destruction of the mRNA, through a complex mechanism involved inRNA-interference. In many of these strategies, mainly oligonucleotidesare used as active agents, although small molecules and other structureshave also been applied. While oligonucleotide-based strategies formodulating gene expression have a huge potential for the treatment ofsome cancers, pharmacological applications of oligonucleotides have beenhindered mainly by ineffective delivery of these compounds to theirsites of action within cancer cells. (P. Herdewijn et al., AntisenseNucleic Acids Drug Dev., 2000, 10: 297-310; Y. Shoji and H. Nakashima,Curr. Charm. Des., 2004, 10: 785-796; A. W Tong et al., Curr. Opin. Mol.Ther., 2005, 7: 114-124).

In certain embodiments, provided chlorotoxin conjugates comprise areduced lysine chlorotoxin polypeptide and a nucleic acid molecule thatis useful as a therapeutic (e.g., anti-cancer) agent. A variety ofchemical types and structural forms of nucleic acid can be suitable forsuch strategies. These include, by way of non-limiting example, DNA,including single-stranded (ssDNA) and double-stranded (dsDNA); RNA,including, but not limited to ssRNA, dsRNA, tRNA, mRNA, rRNA, enzymaticRNA; RNA:DNA hybrids, triplexed DNA (e.g., dsDNA in association with ashort oligonucleotide), and the like.

In some embodiments, the nucleic acid agent is between about 5 and 2000nucleotides long. In some embodiments, the nucleic acid agent is atleast about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides long. Insome embodiments, the nucleic acid agent is less than about 2000, 1900,1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700,600, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 45, 40, 35, 30,25, 20 or fewer nucleotides long.

In some embodiments, the nucleic acid agent comprises a promoter and/orother sequences that regulate transcription. In some embodiments, thenucleic acid agent comprises an origin of replication and/or othersequences that regulate replication. In some embodiments, the nucleicacid agent does not include a promoter and/or an origin of replication.

Nucleic acid anti-cancer agents suitable for use in the practice of thepresent invention include those agents that target genes associated withtumorigenesis and cell growth or cell transformation (e.g.,proto-oncogenes, which code for proteins that stimulate cell division),angiogenic/anti-angiogenic genes, tumor suppressor genes (which code forproteins that suppress cell division), genes encoding proteinsassociated with tumor growth and/or tumor migration, and suicide genes(which induce apoptosis or other forms of cell death), especiallysuicide genes that are most active in rapidly dividing cells.

Examples of genes associated with tumorigenesis and/or celltransformation include MLL fusion genes, BCR-ABL, TEL-AML1, EWS-FLI1,TLS-FUS, PAX3-FKHR, Bcl-2, AML1-ETO, AML1-MTG8, Ras, Fos PDGF, RET, APC,NF-1, Rb, p53, MDM2 and the like; overexpressed genes such as multidrugresistance genes; cyclins; beta-Catenin; telomerase genes; c-myc, n-myc,Bcl-2, Erb-B1 and Erb-B2; and mutated genes such as Ras, Mos, Raf, andMet. Examples of tumor suppressor genes include, but are not limited to,p53, p21, RB1, WT1, NF1, VHL, APC, DAP kinase, p16, ARF, Neurofibromin,and PTEN. Examples of genes that can be targeted by nucleic acid agentsuseful in anti-cancer therapy include genes encoding proteins associatedwith tumor migration such as integrins, selectins, andmetalloproteinases; anti-angiogenic genes encoding proteins that promoteformation of new vessels such as Vascular Endothelial Growth Factor(VEGF) or VEGFr; anti-angiogenic genes encoding proteins that inhibitneovascularization such as endostatin, angiostatin, and VEGF-R2; andgenes encoding proteins such as interleukins, interferon, fibroblastgrowth factor (α-FGF and (β-FGF), insulin-like growth factor (e.g.,IGF-1 and IGF-2), Platelet-derived growth factor (PDGF), tumor necrosisfactor (TNF), Transforming Growth Factor (e.g., TGF-α and TGF-β,Epidermal growth factor (EGF), Keratinocyte Growth Factor (KGF), stemcell factor and its receptor c-Kit (SCF/c-Kit) ligand, CD40L/CD40, VLA-4VCAM-1, ICAM-1/LFA-1, hyalurin/CD44, and the like. As will be recognizedby one skilled in the art, the foregoing examples are not exclusive.

Nucleic acid agents suitable for use in the invention may have any of avariety of uses including, for example, use as anti-cancer or othertherapeutic agents, probes, primers, etc. Nucleic acid agents may haveenzymatic activity (e.g., ribozyme activity), gene expression inhibitoryactivity (e.g., as antisense or siRNA agents, etc), and/or otheractivities. Nucleic acids agents may be active themselves or may bevectors that deliver active nucleic acid agents (e.g., throughreplication and/or transcription of a delivered nucleic acid). Forpurposes of the present specification, such vector nucleic acids areconsidered “therapeutic agents” if they encode or otherwise deliver atherapeutically active agent, even if they do not themselves havetherapeutic activity.

In certain embodiments, chlorotoxin conjugates comprise a nucleic acidtherapeutic agent that comprises or encodes an antisense compound. Theterms “antisense compound or agent,” “antisense oligomer,” “antisenseoligonucleotide,” and “antisense oligonucleotide analog” are used hereininterchangeably, and refer to a sequence of nucleotide bases and asubunit-to-subunit backbone that allows the antisense compound tohybridize to a target sequence in an RNA by Watson-Crick base pairing toform an RNA oligomer heteroduplex within the target sequence. Theoligomer may have exact sequence complementarity within the targetsequence or near complementarity. Such antisense oligomers may block orinhibit translation of the mRNA containing the target sequence, orinhibit gene transcription. Antisense oligomers may bind todouble-stranded or single-stranded sequences.

Examples of antisense oligonucleotides suitable for use in the practiceof the present invention include, for example, those mentioned in thefollowing reviews: R. A Stahel et al., Lung Cancer, 2003, 41: S81-S88;K. F. Pirollo et al., Pharmacol. Ther., 2003, 99: 55-77; A. C. Stephensand R. P. Rivers, Curr. Opin. Mol. Ther., 2003, 5: 118-122; N. M. Deanand C. F. Bennett, Oncogene, 2003, 22: 9087-9096; N. Schiavone et al.,Curr. Pharm. Des., 2004, 10: 769-784; L. Vidal et al., Eur. J. Cancer,2005, 41: 2812-2818; T. Aboul-Fadi, Curr. Med. Chem., 2005, 12:2193-2214; M. E. Gleave and B. P. Monia, Nat. Rev. Cancer, 2005, 5:468-479; Y. S. Cho-Chung, Curr. Pharm. Des., 2005, 11: 2811-2823; E.Rayburn et al., Lett. Drug Design & Discov., 2005, 2: 1-18; E. R.Rayburn et al., Expert Opin. Emerg. Drugs, 2006, 11: 337-352; I. Tammand M. Wagner, Mol. Biotechnol., 2006, 33: 221-238 (each of which isincorporated herein by reference in its entirety).

Examples of suitable antisense oligonucleotides include, for exampleoblimersen sodium (also known as GENASENSE® or G31239, developed byGenta, Inc., Berkeley Heights, N.J.), a phosphorothioate oligomertargeted towards the initiation codon region of the bcl-2 mRNA. Bcl-2 isa potent inhibitor of apoptosis and is overexpressed in many cancerincluding follicular lymphomas, breast cancer, colon cancer, prostatecancer, and intermediate/high-grade lymphomas (C. A. Stein et al.,Semin. Oncol., 2005, 32: 563-573; S. R. Frankel, Semin. Oncol., 2003,30: 300-304). Other suitable antisense oligonucleotides include GEM-231(HYB0165, Hybridon, Inc., Cambridge, Mass.), which is a mixed backboneoligonucleotide directed against cAMP-dependent protein kinase A (PKA)(S. Goel et al., Clin. Cancer Res., 203, 9: 4069-4076); AFFINITAK™ (ISIS3521 or aprinocarsen, ISIS pharmaceuticals, Inc., Carlsbad, Calif.), anantisense inhibitor of PKCalpha; OGX-011 (Isis 112989, IsisPharmaceuticals, Inc.), a 2′-methoxyethyl modified antisenseoligonucleotide against clusterin, a glycoprotein implicated in theregulation of the cell cycle, tissue remodeling, lipid transport, andcell death and which is overexpressed in cancers of breast, prostate andcolon; ISIS 5132 (Isis 112989, Isis Pharmaceuticals, Inc.), aphosphorothioate oligonucleotide complementary to a sequence of the3′-unstranslated region of the c-raf-1 mRNA (S. P. Henry et al.,Anticancer Drug Des., 1997, 12: 409-420; B. P. Monia et al., Proc. Natl.Acad. Sci. USA, 1996, 93: 15481-15484; C. M. Rudin et al., Clin. CancerRes., 2001, 7: 1214-1220); ISIS 2503 (Isis Pharmaceuticals, Inc.), aphosphorothioate oligonucleotide antisense inhibitor of human H-ras mRNAexpression (J. Kurreck, Eur. J. Biochem., 2003, 270: 1628-1644);oligonucleotides targeting the X-linked inhibitor of apoptosis protein(XIAP), which blocks a substantial portion of the apoptosis pathway,such as GEM 640 (AEG 35156, Aegera Therapeutics Inc. and Hybridon, Inc.)or targeting survivin, an inhibitor of apoptosis protein (IAP), such asISIS 23722 (Isis Pharmaceuticals, Inc.), a 2′-O-methoxyethyl chimericoligonucleotide; MG98, which targets DNA methyl transferase; andGTI-2040 (Lorus Therapeutics, Inc. Toronto, Canada), a 20-meroligonucleotide that is complementary to a coding region in the mRNA ofthe R2 small subunit component of human ribonucleotide reductase.

Other suitable antisense oligonucleotides include antisenseoligonucleotides that are being developed against Her-2/neu, c-Myb,c-Myc, and c-Raf (see, for example, A. Biroccio et al., Oncogene, 2003,22: 6579-6588; Y. Lee et al., Cancer Res., 2003, 63: 2802-2811; B. Lu etal., Cancer Res., 2004, 64: 2840-2845; K. F. Pirollo et al., Pharmacol.Ther., 2003, 99: 55-77; and A. Rait et al., Ann. N.Y. Acad. Sci., 2003,1002: 78-89).

In certain embodiments, chlorotoxin conjugates of the present inventioncomprise a nucleic acid anti-cancer agent that comprises or encodes aninterfering RNA molecule. The terms “interfering RNA” and “interferingRNA molecule” are used herein interchangeably, and refer to an RNAmolecule that can inhibit or downregulate gene expression or silence agene in a sequence-specific manner, for example by mediating RNAinterference (RNAi). RNA interference (RNAi) is an evolutionarilyconserved, sequence-specific mechanism triggered by double-stranded RNA(dsRNA) that induces degradation of complementary target single-strandedmRNA and “silencing” of the corresponding translated sequences (McManusand Sharp, 2002, Nature Rev. Genet., 2002, 3: 737). RNAi functions byenzymatic cleavage of longer dsRNA strands into biologically active“short-interfering RNA” (siRNA) sequences of about 21-23 nucleotides inlength (Elbashir et al., Genes Dev., 2001, 15: 188). RNA interferencehas emerged as a promising approach for therapy of cancer.

An interfering RNA suitable for use in the practice of the presentinvention can be provided in any of several forms. For example, aninterfering RNA can be provided as one or more of an isolated shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA),or short hairpin RNA (shRNA).

Examples of interfering RNA molecules suitable for use in the presentinvention include, for example, the iRNAs cited in the followingreviews: O. Milhavet et al., Pharmacol. Rev., 2003, 55: 629-648; F. Biet al., Curr. Gene. Ther., 2003, 3: 411-417; P. Y. Lu et al., Curr.Opin. Mol. Ther., 2003, 5: 225-234; I. Friedrich et al., Semin. CancerBiol., 2004, 14: 223-230; M. Izquierdo, Cancer Gene Ther., 2005, 12:217-227; P. Y. Lu et al., Adv. Genet., 2005, 54: 117-142; G. R. Devi,Cancer Gene Ther., 2006, 13: 819-829; M. A. Behlke, Mol. Ther., 2006,13: 644-670; and L. N. Putral et al., Drug News Perspect., 2006, 19:317-324 (the contents of each of which are incorporated herein byreference in their entirety).

Other examples of suitable interfering RNA molecules include, but arenot limited to, p53 interfering RNAs (e.g., T. R. Brummelkamp et al.,Science, 2002, 296: 550-553; M. T. Hemman et al., Nat. Genet., 2003, 33:396-400); interfering RNAs that target the bcr-abl fusion, which isassociated with development of chronic myeloid leukemia and acutelymphoblastic leukemia (e.g., M. Scherr et al., Blood, 2003, 101:1566-1569; M. J. Li et al., Oligonucleotides, 2003, 13: 401-409),interfering RNAs that inhibit expression of NPM-ALK, a protein that isfound in 75% of anaplastic large cell lymphomas and leads to expressionof a constitutively active kinase associated with tumor formation (U.Ritter et al., Oligonucleotides, 2003, 13: 365-373); interfering RNAsthat target oncogenes, such as Raf-1 (T. F. Lou et al.,Oligonucleotides, 2003, 13: 313-324), K-Ras (T. R. Brummelkamp et al.,Cancer Cell, 2002, 2: 243-247), erbB-2 (G. Yang et al., J. Biol. Chem.,2004, 279: 4339-4345); interfering RNAs that target b-catenin protein,whose over-expression leads to transactivation of the T-cell factortarget genes, which is thought to be the main transforming event incolorectal cancer (M. van de Wetering et al., EMBO Rep., 2003, 4:609-615).

In certain embodiments, chlorotoxin conjugates comprise a nucleic acidtherapeutic agent that is a ribozyme. As used herein, the term“ribozyme” refers to a catalytic RNA molecule that can cleave other RNAmolecules in a target-specific manner Ribozymes can be used todownregulate the expression of any undesirable products of genes ofinterest. Examples of ribozymes that can be used in the practice of thepresent invention include, but are not limited to, ANGIOZYME® (RPI.4610,Sima Therapeutics, Boulder, Colo.), a ribozyme targeting the conservedregion of human, mouse, and rat vascular endothelial growth factorreceptor (VEGFR)-1 mRNA, and HERZYME™ (Sima Therapeutics; anti-HumanEpidermal Growth Factor Receptor type 2 (HER2) ribozyme).

Photosensitizers

In certain embodiments, entities or moieties within chlorotoxinconjugates comprise a photosensitizer used in photodynamic therapy(PDT). In PDT, local or systemic administration of a photosensitizer toa patient is followed by irradiation with light that is absorbed by thephotosensitizer in the tissue or organ to be treated. Light absorptionby the photosensitizer generates reactive species (e.g., radicals) thatare detrimental to cells. For maximal efficacy, a photosensitizertypically is in a form suitable for administration, and also in a formthat can readily undergo cellular internalization at the target site,often with some degree of selectivity over normal tissues.

While some photosensitizers (e.g., porfimer sodium) (PHOTOFRIN®), QLT,Inc., Vancouver, BC, Canada) have been delivered successfully as part ofa simple aqueous solution, such aqueous solutions may not be suitablefor hydrophobic photosensitizer drugs, such as those that have a tetra-or poly-pyrrole-based structure. These drugs have an inherent tendencyto aggregate by molecular stacking, which results in a significantreduction in the efficacy of the photosensitization processes (Siggel etal., J. Phys. Chem., 1996, 100: 2070-2075). Approaches to minimizeaggregation include liposomal formulations (e.g., for benzoporphyrinderivative monoacid A, BPDMA, Verteporfin (VISUDYNE®), QLT, Inc.,Vancouver, Canada; and zinc phthalocyanine, CIBA-Geigy, Ltd., Basel,Switzerland), and conjugation of photosensitizers to biocompatible blockcopolymers (Peterson et al., Cancer Res., 1996, 56: 3980-3985) and/orantibodies (Omelyanenko et al., Int. J. Cancer, 1998, 75: 600-608).

Chlorotoxin conjugates comprising a reduced lysine chlorotoxinpolypeptide associated with a photosensitizer can be used as newdelivery systems in PDT. In addition to reducing photosensitizeraggregation, delivery of photosensitizers according to the presentinvention exhibits other advantages such as increased specificity fortarget tissues/organ and cellular internalization of thephotosensitizer.

Photosensitizers suitable for use in the present invention include anyof a variety of synthetic and naturally occurring molecules that havephotosensitizing properties useful in PDT. In certain embodiments, theabsorption spectrum of the photosensitizer is in the visible range,typically between 350 nm and 1200 nm, preferably between 400 nm and 900nm, e.g., between 600 nm and 900 nm. Suitable photosensitizers that canbe coupled to toxins according to the present invention include, but arenot limited to, porphyrins and porphyrin derivatives (e.g., chlorins,bacteriochlorins, isobacteriochlorins, phthalocyanines, andnaphthalocyanines); metalloporphyrins, metallophthalocyanines,angelicins, chalcogenapyrrillium dyes, chlorophylls, coumarins, flavinsand related compounds such as alloxazine and riboflavin, fullerenes,pheophorbides, pyropheophorbides, cyanines (e.g., merocyanine 540),pheophytins, sapphyrins, texaphyrins, purpurins, porphycenes,phenothiaziniums, methylene blue derivatives, naphthalimides, nile bluederivatives, quinones, perylenequinones (e.g., hypericins, hypocrellins,and cercosporins), psoralens, quinones, retinoids, rhodamines,thiophenes, verdins, xanthene dyes (e.g., eosins, erythrosins, rosebengals), dimeric and oligomeric forms of porphyrins, and prodrugs suchas 5-aminolevulinic acid (R. W. Redmond and J. N. Gamlin, Photochem.Photobiol., 1999, 70: 391-475).

Exemplary photosensitizers suitable for use in the present inventioninclude those described in U.S. Pat. Nos. 5,171,741; 5,171,749;5,173,504; 5,308,608; 5,405,957; 5,512,675; 5,726,304; 5,831,088;5,929,105; and 5,880,145 (the contents of each of which are incorporatedherein by reference in their entirety).

Radiosensitizers

In certain embodiments, chlorotoxin conjugates comprise aradiosensitizer. As used herein, the term “radiosensitizer” refers to amolecule, compound or agent that makes tumor cells more sensitive toradiation therapy. Administration of a radiosensitizer to a patientreceiving radiation therapy generally results in enhancement of theeffects of radiation therapy. Ideally, a radiosensitizer exerts itsfunction only on target cells. For ease of use, a radiosensitizer shouldalso be able to find target cells even if it is administeredsystemically. However, currently available radiosensitizers aretypically not selective for tumors, and they are distributed bydiffusion in a mammalian body. Chlorotoxin conjugates of the presentinvention can be used as a new delivery system for radiosensitizers.

A variety of radiosensitizers are known in the art. Examples ofradiosensitizers suitable for use in the present invention include, butare not limited to, paclitaxel (TAXOL®), carboplatin, cisplatin, andoxaliplatin (Amorino et al, Radiat. Oncol. Investig. 1999; 7: 343-352;Choy, Oncology, 1999, 13: 22-38; Safran et al., Cancer Invest., 2001,19: 1-7; Dionet et al., Anticancer Res., 2002, 22: 721-725; Cividalli etal., Radiat. Oncol. Biol. Phys., 2002, 52: 1092-1098); gemcitabine(GEMZAR®) (Choy, Oncology, 2000, 14: 7-14; Mornex and Girard, Annals ofOncology, 2006, 17: 1743-1747); etanidazole (NITROLMIDAZOLE®) (Inanamiet al., Int. J. Radiat. Biol., 2002, 78: 267-274); misonidazole(Tamulevicius et al., Br. J. Radiology, 1981, 54: 318-324; Palcic etal., Radiat. Res., 1984, 100: 340-347), tirapazamine (Masunaga et al.,Br. J. Radiol., 2006, 79: 991-998; Rischin et al., J. Clin. Oncol.,2001, 19: 535-542; Shulman et al., Int. J. Radiat. Oncol. Biol. Phys.,1999, 44: 349-353); and nucleic acid base derivatives, e.g., halogenatedpurines or pyrimidines, such as 5-fluorodeoxyuridine (Buchholz et al.,Int. J. Radiat. Oncol. Biol. Phys., 1995, 32: 1053-1058).

Radioisotopes

In certain embodiments, chlorotoxin conjugates comprise a radioisotope.Examples of suitable radioisotopes include any α-, β- or γ-emitter,which, when localized at a tumor site, results in cell destruction (S.E. Order, “Analysis, Results, and Future Prospective of the TherapeuticUse of Radiolabeled Antibody in Cancer Therapy”, Monoclonal Antibodiesfor Cancer Detection and Therapy, R. W. Baldwin et al. (Eds.), AcademicPress, 1985). Examples of such radioisotopes include, but are notlimited to, iodine-131 (¹³¹I), iodine-125 (¹²⁵I), bismuth-212 (²¹²Bi),bismuth-213 (²¹³Bi), astatine-211 (²¹¹At), rhenium-186 (¹⁸⁶Re),rhenium-188 (¹⁸⁸Re), phosphorus-32 (³²P), yttrium-90 (⁹⁰yY),samarium-153 (¹⁵³Sm), and lutetium-177 (¹⁷⁷Lu).

Superantigens

In certain embodiments, chlorotoxin conjugates comprise a superantigenor biologically active portion thereof. Superantigens constitute a groupof bacterial and viral proteins that are extremely efficient inactivating a large fraction of the T-cell population. Superantigens binddirectly to the major histocompatibility complex (MHC) without beingprocessed. In fact, superantigens bind unprocessed outside theantigen-binding groove on the MHC class II molecules, thereby avoidingmost of the polymorphism in the conventional peptide-binding site.

A superantigen-based tumor therapeutic approach has been developed forthe treatment of solid tumors. In this approach, a targeting moiety, forexample, an antibody or antibody fragment, is conjugated to asuperantigen, providing a targeted superantigen. If the antibody, orantibody fragment, recognizes a tumor-associated antigen, the targetedsuperantigen, bound to tumors cells, can trigger superantigen-activatedcytotoxic T-cells to kill the tumor cells directly bysuperantigen-dependent cell mediated cytotoxicity. (See, e.g., Søgaardet al. (1996) “Antibody-targeted superantigens in cancer immunotherapy,”Immunotechnology, 2(3): 151-162, the entire contents of which are hereinincorporated by reference.)

Superantigen-based tumor therapeutics have had some success. Forexample, fusion proteins with wild-type staphylococcal enterotoxin A(SEA) have been investigated in clinical trials of colorectal andpancreatic cancer (Giantonio et al., J. Clin. Oncol., 1997, 15:1994-2007; Alpaugh et al., Clin. Cancer Res., 1998, 4: 1903-1914; Chenget al., J. Clin. Oncol., 2004, 22: 602-609; the entire contents of eachof which are herein incorporated by reference); staphylococcalsuperantigens of the enterotoxin gene cluster (egc) have been studiedfor the treatment of non-small cell lung cancer (Terman et al., Clin.Chest Med., 2006, 27: 321-324, the entire contents of which are hereinincorporated by reference), and staphylococcal enterotoxin B has beenevaluated for the intravesical immunotherapy of superficial bladdercancer (Perabo et al., Int. J. Cancer, 2005, 115: 591-598, the entirecontents of which are herein incorporated by reference).

A superantigen, or a biologically active portion thereof, can beassociated to a reduced lysine chlorotoxin polypeptide to form achlorotoxin conjugate according to the present invention and used in atherapy, e.g., an anti-cancer therapy, as described herein.

Examples of superantigens suitable for use in the present inventioninclude, but are not limited to, staphylococcal enterotoxin (SE) (e.g.,staphylococcal enterotoxin A (SEA) or staphylococcal enterotoxin E(SEE)), Streptococcus pyogenes exotoxin (SPE), Staphylococcus aureustoxic shock-syndrome toxin (TSST-1), streptococcal mitogenic exotoxin(SME), streptococcal superantigen (SSA), and staphylococcalsuperantigens of the enterotoxin gene cluster. As known to one skilledin the art, the three-dimensional structures of the above listedsuperantigens can be obtained from the Protein Data Bank. Similarly, thenucleic acid sequences and the amino acid sequences of the above listedsuperantigens and other superantigens can be obtained from GenBank.

Prodrug Activating Enzymes

In certain embodiments, a chlorotoxin conjugate of the present inventionmay be used in directed enzyme prodrug therapy. In a directed enzymeprodrug therapy approach, a directed/targeted enzyme and a prodrug areadministered to a subject, wherein the targeted enzyme is specificallylocalized to a portion of the subject's body where it converts theprodrug into an active drug. The prodrug can be converted to an activedrug in one step (by the targeted enzyme) or in more than one step. Forexample, the prodrug can be converted to a precursor of an active drugby the targeted enzyme. The precursor can then be converted into theactive drug by, for example, the catalytic activity of one or moreadditional targeted enzymes, one or more non-targeted enzymesadministered to the subject, one or more enzymes naturally present inthe subject or at the target site in the subject (e.g., a protease,phosphatase, kinase or polymerase), by an agent that is administered tothe subject, and/or by a chemical process that is not enzymaticallycatalyzed (e.g., oxidation, hydrolysis, isomerization, epimerization,etc.).

Different approaches have been used to direct/target the enzyme to thesite of interest. For example, in ADEPT (antibody-directed enzymeprodrug therapy), an antibody designed/developed against a tumor antigenis linked to an enzyme and injected in a subject, resulting in selectivebinding of the enzyme to the tumor. When the discrimination betweentumor and normal tissue enzyme levels is sufficient, a prodrug isadministered to the subject. The prodrug is converted to its active formby the enzyme only within the tumor. Selectivity is achieved by thetumor specificity of the antibody and by delaying prodrug administrationuntil there is a large differential between tumor and normal tissueenzyme levels. Early clinical trials are promising and indicate thatADEPT may become an effective treatment for all solid cancers for whichtumor-associated or tumor-specific antibodies are known. Tumors havealso been targeted with the genes encoding for prodrug activatingenzymes. This approach has been called virus-directed enzyme prodrugtherapy (VDEPT) or more generally GDEPT (gene-directed enzyme prodrugtherapy, and has shown good results in laboratory systems. Otherversions of directed enzyme prodrug therapy include PDEPT(polymer-directed enzyme prodrug therapy), LEAPT (lectin-directedenzyme-activated prodrug therapy), and CDEPT (clostridial-directedenzyme prodrug therapy). A conjugate according to the present invention,which comprises a prodrug activating enzyme associated with a reducedlysine chlorotoxin polypeptide, can be used in a similar way.

Nonlimiting examples of enzyme/prodrug/active drug combinations suitablefor use in the present invention are described, for example, in Bagshaweet al., Current Opinions in Immunology, 1999, 11: 579-583; Wilman,“Prodrugs in Cancer Therapy”, Biochemical Society Transactions, 14:375-382, 615^(th) Meeting, Belfast, 1986; Stella et al., “Prodrugs: AChemical Approach To Targeted Drug Delivery”, in “Directed DrugDelivery”, Borchardt et al., (Eds), pp. 247-267 (Humana Press, 1985).Nonlimiting examples of enzyme/prodrug/active anti-cancer drugcombinations are described, for example, in Rooseboom et al., Pharmacol.Reviews, 2004, 56: 53-102.

Examples of prodrug activating enzymes include, but are not limited to,nitroreductase, cytochrome P450, purine-nucleoside phosphorylase,thymidine kinase, alkaline phosphatase, β-glucuronidase,carboxypeptidase, penicillin amidase, β-lactamase, cytosine deaminase,and methionine γ-lyase.

Examples of anti-cancer drugs that can be formed in vivo by activationof a prodrug by a prodrug activating enzyme include, but are not limitedto, 5-(aziridin-1-yl)-4-hydroxyl-amino-2-nitro-benzamide,isophosphoramide mustard, phosphoramide mustard, 2-fluoro adenine,6-methylpurine, ganciclovir-triphosphate nucleotide, etoposide,mitomycin C, p-[N,N-bis(2-chloroethyl)amino]phenol (POM), doxorubicin,oxazolidinone, 9-aminocamptothecin, mustard, methotrexate, benzoic acidmustard, doxorubicin, adriamycin, daunomycin, caminomycin, bleomycins,esperamicins, melphalan, palytoxin, 4-desacetylvinblastine-3-carboxylicacid hydrazide, phenylenediamine mustard,4′-carboxyphthalato(1,2-cyclohexane-diamine) platinum, taxol,5-fluorouracil, methylselenol, and carbonothionic difluoride.

b. Anti-Angiogenic Agents

In certain embodiments, a therapeutic (e.g., anti-cancer) agent within achlorotoxin conjugate of the present invention comprises ananti-angiogenic agent. Antiangiogenic agents suitable for use in thepresent invention include any molecule, compound, or factor that blocks,inhibits, slows down, or reduces the process of angiogenesis, or theprocess by which new blood vessels form by developing from preexistingvessels. Such a molecule, compound, or factor can block angiogenesis byblocking, inhibiting, slowing down, or reducing any of the stepsinvolved in angiogenesis, including (but not limited to) steps of (1)dissolution of the membrane of the originating vessel, (2) migration andproliferation of endothelial cells, and (3) formation of new vasculatureby migrating cells.

Examples of anti-angiogenic agents include, but are not limited to,bevacizumab (AVASTIN®), celecoxib (CELEBREX®), endostatin, thalidomide,EMD121974 (Cilengitide), TNP-470, squalamine, combretastatin A4,interferon-α, anti-VEGF antibody, SU5416, SU6668, PTK787/2K 22584,Marimistal, AG3340, COL-3, AE-941 (NEOVASTAT™), and BMS-275291.

Anti-angiogenic agents may be used in a variety of therapeutic contexts,including, but not limited to, anti-cancer therapies and therapies formacular degeneration.

As will be recognized by one skilled in the art, the specific examplesof therapeutic agents cited herein represent only a very small number ofthe therapeutic agents that are suitable for use in the practice of thepresent invention.

2. Detectable Entities/Moieties

In certain embodiments, provided conjugates comprise one or moredetectable entities or moieties, i.e., conjugates are “labeled” withsuch entities or moieties. In some such embodiments, such conjugates areuseful in diagnostic applications.

Any of a wide variety of detectable agents can be used in the practiceof the present invention. Suitable detectable agents include, but arenot limited to: various ligands, radionuclides; fluorescent dyes;chemiluminescent agents (such as, for example, acridinum esters,stabilized dioxetanes, and the like); bioluminescent agents; spectrallyresolvable inorganic fluorescent semiconductors nanocrystals (i.e.,quantum dots); microparticles; metal nanoparticles (e.g., gold, silver,copper, platinum, etc.); nanoclusters; paramagnetic metal ions; enzymes;colorimetric labels (such as, for example, dyes, colloidal gold, and thelike); biotin; dioxigenin; haptens; and proteins for which antisera ormonoclonal antibodies are available.

a. Radioactive and/or Paramagnetic Isotopes or Ions

In certain embodiments, a reduced lysine chlorotoxin polypeptide islabeled with a radioactive and/or paramagnetic isotope or ion. Forexample, a reduced lysine chlorotoxin polypeptide may beisotopically-labeled (i.e., may contain one or more atoms that have beenreplaced by an atom having an atomic mass or mass number different fromthe atomic mass or mass number usually found in nature) or an isotopemay be attached to the reduced lysine chlorotoxin polypeptide.Non-limiting examples of isotopes that can be incorporated into reducedlysine chlorotoxin polypeptides include isotopes of hydrogen, carbon,fluorine, phosphorous, copper, gallium, yttrium, technetium, indium,iodine, rhenium, thallium, bismuth, astatine, samarium, and lutetium(i.e., ³H, ³H, ¹³C, ¹⁴C, ¹⁸F, ³²P, ³⁵S, ⁶⁴Cu, ⁶⁷Ga, ⁹⁰Y, ^(99M)Tc,¹¹¹In, ¹²⁵I, ¹²³I, ¹³¹I, ¹³⁵I, ¹⁸⁶Re, ¹⁸⁷Re, ²⁰¹Tl, ²¹²Bi, ²¹¹At, ¹⁵³Sm,¹⁷⁷Lu).

In certain embodiments, reduced lysine chlorotoxin polypeptides comprisea radioisotope that is detectable by Single Photon Emission ComputedTomography (SPECT) or Position Emission Tomography (PET). Examples ofsuch radionuclides include, but are not limited to, iodine-131 (¹³¹I),iodine 125 (¹²⁵I), bismuth-212 (²¹²Bi), bismuth-213 (²¹³Bi),astatine-221 (²²¹At), copper-67 (⁶⁷Cu), copper-64 (⁶⁴Cu), rhenium-186(¹⁸⁶Re) rhenium-188 (¹⁸⁸Re), phosphorus-32 (³²P), samarium-153 (¹⁵³Sm),lutetium-177 (¹⁷⁷Lu), technetium-99m (^(99m)Tc), gallium-67 (⁶⁷Ga),indium-111, (¹¹¹In), and thallium-201 (²⁰¹Tl).

In certain embodiments, a reduced lysine chlorotoxin polypeptide islabeled with a radioisotope that is detectable by Gamma camera. Examplesof such radioisotopes include, but are not limited to, iodine-131(¹³¹I), and technetium-99m (^(99m)Tc).

In certain embodiments, a reduced lysine chlorotoxin polypeptide islabeled with a paramagnetic metal ion that is a good contrast enhancerin Magnetic Resonance Imaging (MRI). Examples of such paramagnetic metalions include, but are not limited to, gadolinium III (Gd3+), chromium111 (Cr3+), dysprosium III (Dy3+), iron 111 (Fe3+), manganese II (Mn2+),and ytterbium III (Yb3+). In certain embodiments, the labeling moietiescomprises gadolinium III (Gd3+). Gadolinium is an FDA-approved contrastagent for MRI, which accumulates in abnormal tissues causing theseabnormal areas to become very bright (enhanced) on the magneticresonance image. Gadolinium is known to provide great contrast betweennormal and abnormal tissues in different areas of the body, inparticular in the brain.

In certain embodiments, a reduced lysine chlorotoxin polypeptide islabeled with a stable paramagnetic isotope detectable by nuclearmagnetic resonance spectroscopy (MRS). Examples of suitable stableparamagnetic isotopes include, but are not limited to, carbon-13 (¹³C)and fluorine-19 (¹⁹F).

In some embodiments, metal isotopes are non-covalently attached to thereduced lysine chlorotoxin conjugate by chelation. Examples of chelationinclude chelation of a metal isotope to a poly-His region fused to areduced lysine chlorotoxin polypeptide.

In some embodiments, a metal such as gadolinium (Gd) is incorporatedinto a reduced lysine chlorotoxin polypeptide either through covalentbonding or through chelation, as described above.

b. Fluorescent Dyes

In certain embodiments, a reduced lysine chlorotoxin polypeptide islabeled with a fluorescent dye. Numerous known fluorescent dyes of awide variety of chemical structures and physical characteristics aresuitable for use in the practice of the present invention. Suitablefluorescent dyes include, but are not limited to, fluorescein andfluorescein dyes (e.g., fluorescein isothiocyanine or FITC,naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxyfluorescein,6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryldyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes(e.g., carboxytetramethyl-rhodamine or TAMRA, carboxyrhodamine 6G,carboxy-X-rhodamine (ROX™), sulforhodamine B (LISSAMINE™ rhodamine B),rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine(TMR), etc.), coumarin and coumarin dyes (e.g., methoxycoumarin,dialkylaminocoumarin, hydroxycoumarin, aminomethylcoumarin (AMCA),etc.), OREGON GREEN™ Dyes (e.g., OREGON GREEN™ 488, OREGON GREEN™ 500,OREGON GREEN™ 514., etc.), TEXAS RED™, TEXAS RED™-X, SPECTRUM RED™,SPECTRUM GREEN™, cyanine dyes (e.g., CY-3™, CY-5™, CY-3.5™, CY-5.5™,etc.), ALEXA FLUOR™ dyes (e.g., ALEXA FLUOR™ 350, ALEXA FLUOR™ 488,ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594,ALEXA FLUOR™ 633, ALEXA FLUOR™ 660, ALEXA FLUOR™ 680, etc.), BODIPY™dyes (e.g., BODIPY™ FL, BODIPY™ R6G, BODIPY™ TMR, BODIPY™ TR, BODIPY™530/550, BODIPY™ 558/568, BODIPY™ 564/570, BODIPY™ 576/589, BODIPY™581/591, BODIPY™ 630/650, BODIPY™ 650/665, etc.), IRDyes (e.g., IRD40,IRD 700, IRD 800, etc.), and the like. For more examples of suitablefluorescent dyes and methods for coupling fluorescent dyes to otherchemical entities such as proteins and peptides, see, for example, “TheHandbook of Fluorescent Probes and Research Products”, 9^(th) Ed.,Molecular Probes, Inc., Eugene, Oreg. Favorable properties offluorescent labeling agents include high molar absorption coefficient,high fluorescence quantum yield, and photostability. In someembodiments, labeling fluorophores exhibit absorption and emissionwavelengths in the visible (i.e., between 400 and 750 nm) rather than inthe ultraviolet range of the spectrum (i.e., lower than 400 nm).

c. Enzymes

In certain embodiments, a reduced lysine chlorotoxin polypeptide islabeled with an enzyme. Examples of suitable enzymes include, but arenot limited to, those used in an ELISA, e.g., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase, etc. Otherexamples include beta-glucuronidase, beta-D-glucosidase, urease, glucoseoxidase, etc. An enzyme may be conjugated to a reduced lysinechlorotoxin polypeptide using a linker group such as a carbodiimide, adiisocyanate, a glutaraldehyde, and the like.

It will be recognized by those of ordinary skill in the art that in someembodiments, a particular non-chlorotoxin entity or moiety may servemore than one purpose. For example, a moiety may have both a therapeuticpurpose and a diagnostic purpose. To give but one example, radioactiveiodine such as ¹³¹I has been used as both a radiolabel and a cytotoxictherapeutic agent within a chlorotoxin conjugate in the treatment of avariety of tumors including malignant glioma.

III. Pharmaceutical Compositions

Chlorotoxin conjugates described herein may be administered per seand/or in the form of a pharmaceutical composition. In some embodiments,provided are pharmaceutical compositions comprising an effective amountof at least one chlorotoxin conjugate and at least one pharmaceuticallyacceptable carrier.

A chlorotoxin conjugate, or a pharmaceutical composition thereof, may beadministered according to the present invention in such amounts and forsuch a time as is necessary or sufficient to achieve at least onedesired result. For example, an inventive pharmaceutical composition canbe administered in such amounts and for such a time that it kills cancercells, reduces tumor size, inhibits tumor growth or metastasis, treatsvarious leukemias, and/or prolongs the survival time of mammals(including humans) with those diseases, or otherwise yields clinicalbenefit.

Pharmaceutical compositions of the present invention may be administeredusing any amount and any route of administration effective for achievingthe desired therapeutic effect.

The exact amount of pharmaceutical composition to be administered willvary from subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the condition, and the like(see below).

The optimal pharmaceutical formulation can be varied depending upon theroute of administration and desired dosage. Such formulations mayinfluence the physical state, stability, rate of in vivo release, andrate of in vivo clearance of the administered compounds.

Pharmaceutical compositions of the present invention may be formulatedin dosage unit form for ease of administration and uniformity of dosage.The expression “unit dosage form”, as used herein, refers to aphysically discrete unit of chlorotoxin conjugate (with or without oneor more additional agents) for the patient to be treated. It will beunderstood, however, that the total daily usage of compositions of thepresent invention will be decided by the attending physician within thescope of sound medical judgment.

After formulation with one or more appropriate physiologicallyacceptable carrier(s) or excipient(s) in a desired dosage,pharmaceutical compositions of the present invention can be administeredto humans or other mammals by any suitable route. Various deliverysystems are known and can be used to administer such compositions,including, tablets, capsules, injectable solutions, etc. Methods ofadministration include, but are not limited to, dermal, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,pulmonary, epidural, ocular, and oral routes. A composition may beadministered by any convenient or otherwise appropriate route, forexample, by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral, mucosa, rectal andintestinal mucosa, etc) and may be administered together with otherbiologically active agents. Administration can be systemic and/or local.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents, and suspending agents. Asterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a non-toxic parenterally acceptablediluent or solvent, for example, as a solution in 2,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solutionor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or di-glycerides. Fatty acids such asoleic acid may also be used in the preparation of injectableformulations. Sterile liquid carriers are useful in sterile liquid fromcompositions for parenteral administration.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use. Liquid pharmaceutical compositions which are sterile solutionsor suspensions can be administered by, for example, intravenous,intramuscular, intraperitoneal or subcutaneous injection. Injection maybe via single push or by gradual infusion (e.g., 30 minute intravenousinfusion). Where necessary, the composition may include a localanesthetic to ease pain at the site of injection.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming micro-encapsuledmatrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations can also be prepared by entrapping the drug in liposomes(also known as lipid vesicles) or microemulsions that are compatiblewith body tissues.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups, elixirs, and pressurized compositions. In additionto the active ingredient (i.e., conjugate), the liquid dosage form maycontain inert diluents commonly used in the art such as, for example,water or other solvent, solubilizing agents and emulsifiers such asethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cotton seed, ground nut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions can alsoinclude adjuvants such as wetting agents, suspending agents,preservatives, sweetening, flavoring, and perfuming agents, thickeningagents, colors, viscosity regulators, stabilizers or osmoregulators.Suitable examples of liquid carriers for oral administration includewater (partially containing additives as above; e.g., cellulosederivatives, such as sodium caboxymethyl cellulose solution), alcohols(including monohydric alcohols and polyhydric alcohols such as glycols)and their derivatives, and oils (e.g., fractionated coconut oil andarachis oil)).

Solid dosage forms for oral administration include, for example,capsules, tablets, pills, powders, and granules. In such solid dosageforms, the active ingredient is mixed with at least one inert,physiologically acceptable excipient or carrier such as sodium citrateor dicalcium phosphate and one or more of: (a) fillers or extenders suchas starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b)binders such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants suchas glycerol; (d) disintegrating agents such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate; (e) solution retarding agents such as paraffin;(f) absorption accelerators such as quaternary ammonium compounds; (g)wetting agents such as, for example, cetyl alcohol and glycerolmonostearate; (h) absorbents such as kaolin and bentonite clay; and (i)lubricants such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof.Additional or alternative excipients suitable for solid formulationsinclude surface modifying agents such as non-ionic and anionic surfacemodifying agents. Representative examples of surface modifying agentsinclude, but are not limited to, poloxamer 188, benzalkonium chloride,calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax,sorbitan esters, colloidal silicon dioxide, phosphates, sodiumdodecylsulfate, magnesium aluminum silicate, and triethanolamine. In thecase of capsules, tablets and pills, the dosage form may also comprisebuffering agents. The amount of solid carrier per solid dosage form willvary widely. In some embodiments, the amount of solid carrier per soliddosage form is from about 25 mg to about 1 g.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings, release controlling coatings and other coatings well known inthe pharmaceutical formulating art. They may optionally containopacifying agents and can also be of a composition such that theyrelease the active ingredient(s) only, or preferentially, in a certainpart of the intestinal tract, optionally, in a delayed manner. Examplesof embedding compositions which can be used include polymeric substancesand waxes.

In certain embodiments, it may be desirable to administer an inventivecomposition locally to an area in need of treatment. This may beachieved, for example, by local infusion during surgery, topicallyapplication, by injection, by means of a catheter, by means ofsuppository, or by means of a skin patch or stent or other implant,among other ways.

Some compositions for topical administration may be formulated as a gel,an ointment, a lotion, or a cream which can include carriers such aswater, glycerol, alcohol, propylene glycol, fatty alcohols,triglycerides, fatty acid esters, or mineral oil. Other topical carriersinclude liquid petroleum, isopropyl palmitate, polyethylene glycol,ethanol (95%), polyoxyethylenemonolaurate (5%) in water, or sodiumlauryl sulfate (5%) in water. Other materials such as antioxidants,humectants, viscosity stabilizers, and similar agents may be added asnecessary. Percutaneous penetration enhancers such as1-dodecylazacycloheptan-2-one (AZONE®) may also be included.

In addition, in certain instances, compositions may be disposed withintransdermal devices placed upon, in, or under the skin. Such devicesinclude patches, implants, and injections which release the compoundonto the skin, by either passive or active release mechanisms.Transdermal administrations include all administrations across thesurface of the body and the inner linings of bodily passage includingepithelial and mucosal tissues. Such administrations may be carried outusing the present compositions in lotions, creams, foams, patches,suspensions, solutions, and suppositories (rectal and vaginal).

Transdermal administration may be accomplished, for example, through useof a transdermal patch containing active ingredient(s) and a carrierthat is non-toxic to the skin, and allows the delivery of at least someof the active ingredient(s) for systemic absorption into the bloodstreamvia the skin. The carrier may take any number of forms such as creamsand ointments, pastes, gels, and occlusive devices. Creams and ointmentsmay be viscous liquid or semisolid emulsions of either the oil-in-wateror water-in-oil type. Pastes comprised of absorptive powders dispersedin petroleum or hydrophilic petroleum containing active ingredient(s)may also be suitable. A variety of occlusive devices may be used torelease active ingredient(s) into the bloodstream such as asemipermeable membrane covering a reservoir containing the activeingredient(s) with or without a carrier, or a matrix containing theactive ingredient.

Suppository formulations may be made from traditional materials,including cocoa butter, with or without the addition of waxes to alterthe suppository's melting point, and glycerin. Water soluble suppositorybases, such as polyethylene glycols of various molecular weights, mayalso be used.

Materials and methods for producing various formulations are known inthe art and may be adapted for practicing the subject invention.

Encapsulating Agents

In some embodiments, compositions provided by the present inventioninclude one or more encapsulating agents. In general, an encapsulatingagent can be any physiologically tolerable agent that can be used toentrap an entity such as a conjugate or a moiety. By “entrapped” it ismeant that the encapsulating agent may encircle or enclose the entity,or an “entrapped” entity may be embedded partially or wholly within thematerial comprising the encapsulating agent.

In some embodiments, the encapsulating agent is part of the moiety (suchas therapeutic moiety), and the reduced lysine chlorotoxin polypeptideis conjugated to the encapsulating agent. In some such embodiments, thereduced lysine chlorotoxin polypeptide is conjugated to the outersurface of the encapsulating agent. In some such embodiments, thereduced lysine chlorotoxin polypeptide is exposed on the environmentexternal to the encapsulating agent. The reduced lysine chlorotoxinpolypeptide may be conjugated to the encapsulating agent by a directinteraction (which may be non-covalent or covalent), or it may beconjugated to the encapsulating agent via a linker.

In some embodiments, the conjugate comprising the reduced lysinechlorotoxin polypeptide and the moiety (e.g., therapeutic moiety) isenclosed by the encapsulating agent. The conjugate may be enclosedpartially or wholly within a space or environment (for example, anaqueous environment) defined and/or created by the encapsulating agent.In some embodiments, the conjugate is at least partially embedded withinthe encapsulating agent. For example, if the encapsulating agentcomprises lipid membranes, the conjugate may be at least partiallyembedded within or among lipid molecules in the membrane. In someembodiments, the conjugate is wholly embedded within the encapsulatingagent.

A variety of types of encapsulating agents are known in the art, as aremethods of using such agents to entrap drugs, biomolecules, and thelike. In certain embodiments, the encapsulating agent comprises a smallparticle having a core and a surface. Such encapsulating agents include,but are not limited to, liposomes, micelles, microparticles,nanoparticles, etc.

Liposomes are typically approximately spherically shaped bilayerstructures or vesicles and comprised of natural or syntheticphospholipid membranes. Liposomes may further comprise other membranecomponents such as cholesterol and protein. The interior core ofliposomes typically contain an aqueous solution. Therapeutic agentsand/or conjugates may be dissolved in the aqueous solution. Aspreviously mentioned, therapeutic agents and conjugates may be embeddedwithin the membrane of the liposome. Liposomes may be especially usefulfor delivering agents such as nucleic acid agents (such as thosedescribed above), including inhibitory RNAs such as siRNAs.

Micelles are similar to liposomes, except they generally form from asingle layer of phospholipids and lack an internal aqueous solution.Reverse micelles that are made to include internal aqueous solution mayalso be used in accordance with the present invention.

In some embodiments, the particle is a microparticle, at least onedimension of which averages to be smaller than about 1 μm. For example,the smallest dimension of the particles can average about 100 nm, about120 nm, about 140 nm, about 160 nm, about 180 nm, about 200 nm, about220 nm, about 240 nm, about 260 nm, about 280 nm, about 300 nm, about320 nm, about 340 nm, about 360 nm, about 380 nm, about 400 nm, about420 nm, about 440 nm, about 460 nm, about 480 nm, about 500 nm, about550 nm, about 600 nm, about 650 nm, about 700 nm, about 750 nm, about800 nm, about 850 nm, about 900 nm, or about 950 nm.

In some embodiments, the particle is a nanoparticle, at least onedimension of which averages to be smaller than about 100 μm. Forexample, the smallest dimension of the particles can average about 1 nm,about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm,about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm,about 19 nm, about 20 nm, about 22 nm, about 24 nm, about 26 nm, about28 nm, about 30 nm, about 32 nm, about 34 nm, about 36 nm, about 38 nm,about 40 nm, about 42 nm, about 44 nm, about 46 nm, about 48 nm, about50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm,about 80 nm, about 85 nm, about 90 nm, about 95 nm, or about 99 nm.

In some embodiments, the core of the particle comprises a materialhaving magnetic resonance activity, which may advantageous in diagnosticand/or therapeutic applications. Materials having magnetic resonanceactivity include metals and their oxides, such as aluminum-cobalt-,indium-, iron-, copper-, germanium-, manganese-, nickel-, tin-,titanium-, palladium-, platinum-, selenium-, silicon-, silver-, zinc-,etc. containing metals.

In some embodiments, therapeutic agents comprise nucleic acids. Nucleicacids may be enclosed wholly within the encapsulating agent. In someembodiments, nucleic acid agents are embedded within the encapsulatingagent. For example, the encapsulating agent may be a liposome and thenucleic agent may be enclosed within the liposome. The nucleic acidagent may be at least partially embedded within the lipid molecules ofthe liposome.

Pharmaceutical Packs or Kits

In another aspect, the present invention provides a pharmaceutical packor kit comprising one or more containers (e.g., vials, ampoules, testtubes, flasks or bottles) containing one or more ingredients of apharmaceutical composition as described herein, allowing administrationof a chlorotoxin conjugate of the present invention.

IV. Methods of Using Chlorotoxin Conjugates

In certain embodiments, provided are methods comprising the step ofadministering a composition comprising a chlorotoxin conjugate asdescribed herein to an individual having or suspected of having a tumor,such that the conjugate binds specifically to the tumor. In someembodiments, such methods are useful in treatment and/or diagnosis ofcancer. In some embodiments, such methods are useful in reducing thelikelihood that the individual will develop a tumor, that one or moretumors in the individual will increase in size, that one or more tumorsin the individual will metastasize, and/or that the cancer will progressby any other measure (such as clinical stage).

In certain embodiments, provided are methods comprising the step ofadministering a composition comprising a chlorotoxin conjugate asdescribed herein to an individual having or suspected of having adisease or condition characterized by aberrant angiogenesis, such thatthe chlorotoxin conjugate reduces extent of angiogenesis. In someembodiments, the chlorotoxin conjugate prevents the formation ofneovasculature. In some embodiments, the chlorotoxin conjugate causesexisting neovasculature to regress.

A. Dosages and Administration

Compositions according to the present invention may be administeredaccording to a regimen consisting of a single dose or a plurality ofdoses over a period of time.

Chlorotoxin conjugates, or pharmaceutical compositions thereof, may beadministered using any administration route effective for achieving thedesired effect (e.g., therapeutic, diagnostic, etc.). In certainembodiments of the invention, chlorotoxin conjugates (or pharmaceuticalcompositions thereof) are delivered systemically. Typical systemicroutes of administration include, but are not limited to, intramuscular,intravenous, pulmonary, and oral routes. Systemic administration mayalso be performed, for example, by infusion or bolus injection, or byabsorption through epithelial or mucocutaneous linings (e.g., oral,mucosa, rectal and intestinal mucosa, etc). In certain embodiments, thechlorotoxin conjugate is administered intravenously.

Alternatively or additionally, other routes of administration may alsobe used. In certain embodiments, the chlorotoxin conjugate isadministered by a route selected from the group consisting ofintravenous, intracranial (including intracavitary), intramuscular,intratumoral, subcutaneous, intraocular, periocular, topicalapplication, or by combinations thereof.

As discussed below, it may be desirable to reduce extent of angiogenesisin ocular neovascularization diseases. In some embodiments, chlorotoxinconjugates may be delivered to the eye. Delivery to the eye may beachieved, for example, using intraocular and/or periocular routes suchas intravitreal injection, subjunctival injection, etc. Topicalapplication of chlorotoxin agents to the eye may also be achieved, forexample, using eye drops.

Ocular routes of administration may be particularly useful for treatmentof ocular neovascularization diseases such as macular degeneration.

Administration may be one or multiple times daily, weekly (or at someother multiple day interval) or on an intermittent schedule. Forexample, a composition may be administered one or more times per day ona weekly basis for a period of weeks (e.g., 4-10 weeks). Alternatively,a composition may be administered daily for a period of days (e.g., 1-10days) following by a period of days (e.g., 1-30 days) withoutadministration, with that cycle repeated a given number of times (e.g.,2-10 cycles). In some embodiments, at least two, at least three, atleast four, at least five, or at least six doses are administered. Insome embodiments, the composition is administered weekly for at leasttwo weeks, three weeks, four weeks, five weeks, or six weeks.

Administration may be carried out in any convenient manner, or in anycombination of manners, such as by injection (subcutaneous, intravenous,intramuscular, intraperitoneal, or the like), oral administration,and/or intracavitary administration.

Depending on the route of administration, effective doses may becalculated according to the organ function, body weight, or body surfacearea of the subject to be treated. Optimization of the appropriatedosages can readily be made by one skilled in the art in light ofpharmacokinetic data observed in human clinical trials. Final dosageregimen may be determined by the attending physician, consideringvarious factors that modify the action of the drugs, e.g., the drug'sspecific activity, the severity of the damage and the responsiveness ofthe patient, the age, condition, body weight, sex and diet of thepatient, the severity of any present infection, time of administration,the use (or not) of concomitant therapies, and other clinical factors.

Typical dosages range from about 1.0 pg/kg body weight to about 100mg/kg body weight. (Dosages are presented herein in terms of the weightof the reduced lysine chlorotoxin polypeptide part of the conjugate.)

For example, for systemic administration, typical dosages range fromabout 100.0 ng/kg body weight to about 10.0 mg/kg body weight. Forexample, in certain embodiments where a chlorotoxin conjugate isadministered intravenously, dosing of the agent may compriseadministration of one or more doses comprising about 0.001 mg/kg toabout 5 mg/kg, e.g., from about 0.001 mg/kg to about 5 mg/kg, from about0.01 mg/kg to about 4 mg/kg, from about 0.02 mg/kg to about 3 mg/kg,from about 0.03 mg/kg to about 2 mg/kg or from about 0.03 mg/kg to about1.5 mg/kg of chlorotoxin. For example, in some embodiments, one or moredoses of chlorotoxin conjugate may be administered that each containsabout 0.002 mg/kg, about 0.004 mg/kg, about 0.006 mg/kg, about 0.008mg/kg, about 0.009 mg/kg, about 0.01 mg/kg, about 0.02 mg/kg or morethan 0.02 mg/kg of chlorotoxin. In some embodiments, one or more dosesof chlorotoxin conjugate may be administered that each contains about0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about0.07 mg/kg, about 0.09 mg/kg, about 1.0 mg/kg or more than 1.0 mg/kg ofchlorotoxin. In some embodiments, one or more doses of chlorotoxinconjugate may be administered that each contains about 0.05 mg/kg, about0.10 mg/kg, about 0.15 mg/kg, about 0.20 mg/kg, about 0.25 mg/kg, about0.30 mg/kg, about 0.35 mg/kg, about 0.40 mg/kg, about 0.45 mg/kg, about0.50 mg/kg, about 0.55 mg/kg, about 0.60 mg/kg, about 0.65 mg/kg, about0.70 mg/kg, about 0.75 mg/kg, about 0.80 mg/kg, about 0.85 mg/kg, about0.90 mg/kg, about 0.95 mg/kg, about 1.0 mg/kg, or more than about 1mg/kg of chlorotoxin. In yet other embodiments, one or more doses ofchlorotoxin conjugate may be administered that each contains about 1.0mg/kg, about 1.05 mg/kg, about 1.10 mg/kg, about 1.15 mg/kg, about 1.20mg/kg, about 1.25 mg/kg, about 1.3 mg/kg, about 1.35 mg/kg, about 1.40mg/kg, about 1.45 mg/kg, about 1.50 mg/kg, or more than about 1.50 mg/kgof chlorotoxin. In such embodiments, at treatment may compriseadministration of a single dose of chlorotoxin conjugate oradministration of 2 doses, 3 doses, 4 doses, 5 doses, 6 doses or morethan 6 doses. Two consecutive doses may be administered at 1 dayinterval, 2 days interval, 3 days interval, 4 days interval, 5 daysinterval, 6 days interval, 7 days interval, or more than 7 days interval(e.g., 10 days, 2 weeks, or more than 2 weeks).

For direct administration to the site via microinfusion, typical dosagesrange from about 1 ng/kg body weight to about 1 mg/kg body weight.

In certain embodiments where the chlorotoxin conjugate is administeredlocally, in particular in cases of intracavitary administration to thebrain, dosing of the conjugate may comprise administration of one ormore doses comprising about 0.01 mg to about 100 mg of chlorotoxinpolypeptide, e.g., from about 0.05 to about 50 mg, from about 0.1 mg toabout 25 mg, from about 0.1 mg to about 10 mg, from about 0.1 mg toabout 5 mg, or from about 0.1 mg to about 1.0 mg. For example, incertain embodiments, one or more doses of chlorotoxin conjugate may beadministered that each contains about 1 mg, about 1.5 mg, about 2 mg,about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg orabout 5 mg of reduced lysine chlorotoxin polypeptide. In someembodiments, one or more doses of chlorotoxin conjugate may beadministered that each contains about 0.1 mg, about 0.15 mg, about 0.2mg, about 0.25 mg, about 0.3 mg, about 0.35 mg, about 0.4 mg, about 0.45mg, about 0.5 mg, about 0.55 mg, about 0.6 mg, about 0.65 mg, about 0.7mg, about 0.75 mg, about 0.8 mg, about 0.85 mg, about 0.9 mg, about 0.95mg or about 1 mg of reduced lysine chlorotoxin polypeptide. In someembodiments, a treatment may comprise administration of a single dose ofchlorotoxin conjugate or administration of 2 doses, 3 doses, 4 doses, 5doses, 6 doses or more than 6 doses. Two consecutive doses may beadministered at 1 day interval, 2 days interval, 3 days interval, 4 daysinterval, 5 days interval, 6 days interval, 7 days interval, or morethan 7 days interval (e.g., 10 days, 2 weeks, or more than 2 weeks). Insome embodiments, multiple doses are administered, and the amount ofreduced lysine chlorotoxin polypeptide administered is not the same forevery dose. For example, in some embodiments, doses may be adjusted(e.g., escalated or reduced) from one dose to another as determined bythe attending clinician.

It will be appreciated that pharmaceutical combinations of the presentinvention can be employed in combination with additional therapies(i.e., a treatment according to the present invention can beadministered concurrently with, prior to, or subsequently to one or moredesired therapeutics or medical procedures). The particular combinationof therapies (therapeutics and/or procedures) to employ in such acombination regimen will take into account compatibility of the desiredtherapeutics and/or procedures and the desired therapeutic effect to beachieved.

For example, methods and compositions of the present invention can beemployed together with other procedures including surgery, radiotherapy(e.g., γ-radiation, proton beam radiotherapy, electron beamradiotherapy, proton therapy, brachytherapy, and systemic radioactiveisotopes), endocrine therapy, hyperthermia and cryotherapy.

Alternatively or additionally, methods and compositions of the presentinvention can be employed together with other agents to attenuate anyadverse effects (e.g., antiemetics), and/or with other approvedchemotherapeutic drugs, including, but not limited to, alkylating drugs(mechlorethamine, chlorambucil, cyclophosphamide, melphalan,ifosfamide), antimetabolites (methotrexate), purine antagonists andpyrimidine antagonists (6 mercaptopurine, 5 fluorouracil, cytarabile,gemcitabine), spindle poisons (vinblastine, vincristine, vinorelbine,paclitaxel), podophyllotoxins (etoposide, irinotecan, topotecan),antibiotics (doxorubicin, bleomycin, mitomycin), nitrosoureas(carmustine, lomustine), inorganic ions (cisplatin, carboplatin),enzymes (asparaginase), and hormones (tamoxifen, leuprolide, flutamide,and megestrol), to name a few. For a more comprehensive discussion ofupdated cancer therapies see, <http://www.cancer.gov/>, a list of theFDA approved oncology drugs at<http://www.fda.gov/cder/cancer/druglistframe.htm>, and The MerckManual, Seventeenth Ed. 1999, the entire contents of which are herebyincorporated by reference.

Methods and compositions of the present invention can also be employedtogether with one or more further combinations of cytotoxic agents aspart of a treatment regimen. In some embodiments, the furthercombination of cytotoxic agents is selected from: CHOPP(cyclophosphamide, doxorubicin, vincristine, prednisone, andprocarbazine); CHOP (cyclophosphamide, doxorubicin, vincristine, andprednisone); COP (cyclophosphamide, vincristine, and prednisone);CAP-BOP (cyclophosphamide, doxorubicin, pro carbazine, bleomycin,vincristine, and prednisone); m-BACOD (methotrexate, bleomycin,doxorubicin, cyclophosphamide, vincristine, dexamethasone, andleucovorin); ProMACE-MOPP (prednisone, methotrexate, doxorubicin,cyclophosphamide, etoposide, leucovorin, mechloethamine, vincristine,prednisone, and procarbazine); ProMACE-CytaBOM (prednisone,methotrexate, doxorubicin, cyclophosphamide, etoposide, leucovorin,cytarabine, bleomycin, and vincristine); MACOP-B (methotrexate,doxorubicin, cyclophosphamide, vincristine, prednisone, bleomycin, andleucovorin); MOPP (mechloethamine, vincristine, prednisone, andprocarbazine); ABVD (adriamycin/doxorubicin, bleomycin, vinblastine, anddacarbazine); MOPP (mechloethamine, vincristine, prednisone andprocarbazine) alternating with ABV (adriamycin/doxorubicin, bleomycin,and vinblastine); MOPP (mechloethamine, vincristine, prednisone, andprocarbazine) alternating with ABVD (adriamycin/doxorubicin, bleomycin,vinblastine, and dacarbazine); Ch1VPP (chlorambucil, vinblastine,procarbazine, and prednisone); IMVP-16 (ifosfamide, methotrexate, andetoposide); MIME (methyl-gag, ifosfamide, methotrexate, and etoposide);DHAP (dexamethasone, high-dose cytaribine, and cisplatin); ESHAP(etoposide, methylpredisolone, high-dose cytarabine, and cisplatin);CEPP(B) (cyclophosphamide, etoposide, procarbazine, prednisone, andbleomycin); CAMP (lomustine, mitoxantrone, cytarabine, and prednisone);CVP-1 (cyclophosphamide, vincristine, and prednisone), ESHOP (etoposide,methylpredisolone, high-dose cytarabine, vincristine and cisplatin);EPOCH (etoposide, vincristine, and doxorubicin for 96 hours with bolusdoses of cyclophosphamide and oral prednisone), ICE (ifosfamide,cyclophosphamide, and etoposide), CEPP(B) (cyclophosphamide, etoposide,procarbazine, prednisone, and bleomycin), CHOP-B (cyclophosphamide,doxorubicin, vincristine, prednisone, and bleomycin), CEPP-B(cyclophosphamide, etoposide, procarbazine, and bleomycin), and P/DOCE(epirubicin or doxorubicin, vincristine, cyclophosphamide, andprednisone).

B. Indications

Compositions and methods of the present invention can be used in avariety of antiproliferative and/or antiangiogenic contexts to treatand/or diagnose diseases or conditions.

1. Anti-Proliferative Contexts

In certain embodiments, compositions and methods of the presentinvention are used to treat and/or diagnose conditions involvinguncontrolled cell proliferation, such as primary and/or metastaticcancers, and other cancerous conditions. For example, compositions andmethods of the present invention should be useful for reducing size ofsolid tumors, inhibiting tumor growth or metastasis, treating variouslymphatic cancers, and/or prolonging the survival time of mammals(including humans) suffering from these diseases.

Examples of cancers and cancer conditions that can be treated and/ordiagnosed according to the present invention include, but are notlimited to, tumors of the brain and central nervous system (e.g., tumorsof the meninges, brain, spinal cord, cranial nerves and other parts ofthe CNS, such as glioblastomas or medulloblastomas); head and/or neckcancer, breast tumors, tumors of the circulatory system (e.g., heart,mediastinum and pleura, and other intrathoracic organs, vascular tumors,and tumor-associated vascular tissue); tumors of the blood and lymphaticsystem (e.g., Hodgkin's disease, Non-Hodgkin's disease lymphoma,Burkitt's lymphoma, AIDS-related lymphomas, malignantimmunoproliferative diseases, multiple myeloma, and malignant plasmacell neoplasms, lymphoid leukemia, myeloid leukemia, acute or chroniclymphocytic leukemia, monocytic leukemia, other leukemias of specificcell type, leukemia of unspecified cell type, unspecified malignantneoplasms of lymphoid, haematopoietic and related tissues, such asdiffuse large cell lymphoma, T-cell lymphoma or cutaneous T-celllymphoma); tumors of the excretory system (e.g., kidney, renal pelvis,ureter, bladder, and other urinary organs); tumors of thegastrointestinal tract (e.g., esophagus, stomach, small intestine,colon, colorectal, rectosigmoid junction, rectum, anus, and anal canal);tumors involving the liver and intrahepatic bile ducts, gall bladder,and other parts of the biliary tract, pancreas, and other digestiveorgans; tumors of the oral cavity (e.g., lip, tongue, gum, floor ofmouth, palate, parotid gland, salivary glands, tonsil, oropharynx,nasopharynx, puriform sinus, hypopharynx, and other sites of the oralcavity); tumors of the reproductive system (e.g., vulva, vagina, Cervixuteri, uterus, ovary, and other sites associated with female genitalorgans, placenta, penis, prostate, testis, and other sites associatedwith male genital organs); tumors of the respiratory tract (e.g., nasalcavity, middle ear, accessory sinuses, larynx, trachea, bronchus andlung, such as small cell lung cancer and non-small cell lung cancer);tumors of the skeletal system (e.g., bone and articular cartilage oflimbs, bone articular cartilage and other sites); tumors of the skin(e.g., malignant melanoma of the skin, non-melanoma skin cancer, basalcell carcinoma of skin, squamous cell carcinoma of skin, mesothelioma,Kaposi's sarcoma); and tumors involving other tissues includingperipheral nerves and autonomic nervous system, connective and softtissue, retroperitoneoum and peritoneum, eye and adnexa, thyroid,adrenal gland, and other endocrine glands and related structures,secondary and unspecified malignant neoplasms of lymph nodes, secondarymalignant neoplasm of respiratory and digestive systems and secondarymalignant neoplasms of other sites.

In some embodiments, the tumor is cutaneous or intraocular melanoma. Insome embodiments, the tumor is metastatic melanoma. In some embodiments,the tumor is non-small cell lung cancer. In some embodiments, the tumoris colon or colorectal cancer.

In some embodiments, compositions and methods are useful in thetreatment and/or diagnosis of neuroectodermal tumors. (See, e.g., U.S.Pat. No. 6,667,156; the entire contents of which are herein incorporatedby reference.) In some embodiments, the neuroectodermal tumor is glioma.(See, e.g., U.S. Pat. Nos. 5,905,027; 6,028,174; 6,319,891; 6,429,187;and 6,870,029; and International Patent Application publicationsWO03/101475A2, WO09/021,136A1, and WO 2009/140599; the entire contentsof each of which are herein incorporated by reference.) Types of gliomafor which compositions and methods of the invention are useful include,but are not limited to, glioblastoma multiformes (WHO grad IV),anaplastic astrocytomas (WHO grade III), low grade gliomas (WHO gradeII), pliocytic astrocytomas (WHO grade I), oligodendrogliomas,gangliomas, meningiomas, and ependymomas. In some embodiments, theneuroectodermal tumor is selected from the group consisting ofmedulloblastomas, neuroblastomas, pheochromocytomas, melanomas,peripheral primitive neuroectodermal tumors, small cell carcinoma of thelung, Ewing's sarcoma, and metastatic tumors in the brain.

In certain embodiments of the present invention, compositions andmethods are used in the treatment and/or diagnosis of sarcomas. In someembodiments, compositions and methods of the present invention are usedin the treatment and/or diagnosis of bladder cancer, breast cancer,chronic lymphoma leukemia, head and neck cancer, endometrial cancer,Non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer,pancreatic cancer, and prostate cancer. In some embodiments, the sarcomais selected from the group consisting of prostate cancer or breastcancer. (See, e.g., International Patent Application publicationsWO03/101474A1, WO03/10475A2, and WO 2009/140599, the entire contents ofeach of which are herein incorporated by reference.) In someembodiments, the sarcome is pancreatic cancer.

In certain embodiments of the present invention, compositions andmethods are useful in the treatment and/or diagnosis ofmyeloproliferative disorders (e.g., tumors of myeloid origin) and/orlymphoproliferative disorders (e.g., tumors of lymphoid origin). (See,e.g., International Patent Application publication WO05/099774, theentire contents of which are herein incorporated by reference.)

Types of myeloproliferative disorders for which compositions and methodsof the present invention are useful include, but are not limited to,polycythemia vera (PV), essential thrombocythemia (ET), agnogenicmyeloid metaplasia (AMM) (also referred to as idiopathic myelofibrosis(IMF)), and chronic myelogenous leukemia (CML).

In some embodiments, compositions and methods of the present inventionare used to treat and/or diagnose a lymphoproliferative disorder. Insome embodiments, the lymphoproliferative disorder is a non-Hodgkin'slymphoma. In some embodiments, the lymphoproliferative disorder is a Bcell neoplasm, such as, for example, a precursor B-cell lymphoblasticleukemia/lymphoma or a mature B cell neoplasm. Non-limiting types ofmature B cell neoplasms include B cell chronic lymphocyticleukemia/small lymphocytic lymphoma, B cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, splenic marginal zone B cell lymphoma, hairycell leukemia, extranodal marginal zone B cell lymphoma, mantle celllymphoma, follicular lymphoma, nodal marginal zone lymphoma, diffuselarge B cell lymphoma, Burkitt's lymphoma, plasmacytoma, and plasma cellmyeloma.

In some embodiments, compositions and methods of the present inventionare used to treat a T cell neoplasm. Non-limiting types of T cellneoplasms include T cell prolymphocytic leukemia, T cell large granularlymphocytic leukemia, NK cell leukemia, extranodal NK/T cell lymphoma,mycosis fungoides, primary cutaneous anaplastic large cell lymphoma,subcutaneous panniculitis-like T cell lymphoma, enteropathy-typeintestinal T cell lymphoma, hepatosplenic gamma-delta T cell lymphoma,angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma,anaplastic large cell lymphoma, and adult T cell lymphoma.

Tumors that can be treated using compositions and methods of the presentinvention may be refractory to treatment with other chemotherapeutics.The term “refractory”, when used herein in reference to a tumor meansthat the tumor (and/or metastases thereof), upon treatment with at leastone chemotherapeutic other than an inventive composition, shows no oronly weak anti-proliferative response (i.e., no or only weak inhibitionof tumor growth) after the treatment of such a chemotherapeuticagent—that is, a tumor that cannot be treated at all or only withunsatisfying results with other (preferably standard) chemotherapeutics.The present invention, where treatment of refractory tumors and the likeis mentioned, is to be understood to encompass not only (i) tumors whereone or more chemotherapeutics have already failed during treatment of apatient, but also (ii) tumors that can be shown to be refractory byother means, e.g., biopsy and culture in the presence ofchemotherapeutics.

2. Anti-Angiogenic Contexts

Chlorotoxin has been shown to exert anti-angiogenic properties. See,e.g., International Patent Application publication WO2009/117018, theentire contents of which are herein incorporated by reference. Incertain embodiments, compositions and methods of the present inventionare used to treating, diagnose, and/or ameliorate a disease or conditionsuch as, for example cancer (including metastatic cancer, as describedabove), ocular neovascularization (such as macular degeneration),inflammatory diseases (such as arthritis), etc. In some embodiments, thecondition or disease is characterized by choroidal neovascularization.Examples of such conditions or diseases include, but are not limited to,macular degeneration (including wet macular degeneration, age-relatedmacular degeneration, etc.), myopia, ocular trauma, pseudoxanthomaelasticum, and combinations thereof.

Macular degeneration is the leading cause of vision loss and blindnessin Americans aged 65 and older. Macular degeneration typically occurs inthe age-related form (often called AMD or ARMD), though juvenile maculardegeneration occurs as well. In AMD/ARMD, the macula—the part of theretina that is responsible for sharp, central vision—degenerates.Macular degeneration is typically diagnosed as either dry(non-neovascular) or wet (neovascular).

In dry macular degeneration, yellowish spots known as drusen begin toaccumulate from deposits or debris from deteriorating tissue from mostlyaround the macula. Central vision less usually occurs gradually and isnot as severe as vision loss in wet macular degeneration.

Wet macular degeneration, as the “neovascular” designation suggests, ischaracterized by new blood vessels growing aberrantly, e.g., on themacula. Such new blood vessels may grow beneath the retina, leakingblood and fluid. Such leakage causes permanent damage to light-sensitiveretinal cells, which die and create blind spots in central vision. Wetmacular degeneration may be further grouped into two categories. In theoccult form of wet macular degeneration, new blood vessel growth beneaththe retina is not as pronounced and leakage is less evident, typicallyresulting in less severe vision less. In the classic form of wet maculardegeneration, blood vessel growth and scarring have very clear,delineated outlines that are observable beneath the retina. Classic wetmacular degeneration is also known as classic choroidalneovascularization and usually results in more severe vision loss.

Given the role of angiogenesis in wet macular degeneration, whichcomprises many AMD/ARMD cases, inventive compositions and methods may beuseful in treating, diagnosing, and/or ameliorating such disorders.Current therapies for wet macular degeneration involve angiogenesisinhibitors such as ranibizumab (LUCENTIS®), pegaptanib (MACUGEN®),and/or verteporfin (VISUDYNE®), optionally combined with photodynamictherapy (PDT) to target drugs to specific cells. Photocoagulation, inwhich a high energy laser beam is used to create small burns in areas ofthe retina with abnormal blood vessels, is also used to treat wetmacular degeneration.

In some embodiments, chlorotoxin conjugates (or a pharmaceuticalcomposition thereof) are administered to a subject suffering from wetmacular degeneration and/or age-related macular degeneration. Amongsubjects suffering from wet macular degeneration, subjects may sufferfrom the occult or the classic form. In some embodiments, chlorotoxinconjugates cause regression of existing neovasculature. In someembodiments, chlorotoxin conjugates prevent sprouting of new vessels. Incertain embodiments, chlorotoxin conjugates are combined with othertreatments for wet macular degeneration, such as photocoagulation,treatment with other angiogenesis inhibitors, photodynamic therapy, etc.

In some embodiments, chlorotoxin agents as described herein areadministered in combination with or as part of a therapeutic regimenwith one or more therapeutic regimens recommended for treatment of adisease, disorder, or condition associated with angiogenesis. To givebut a few examples, recommended regimens for treatment of cancer can befound at the web site have a URL of www.cancer.gov, the website of theNational Cancer Institute. Recommended regimens for treatment of maculardegeneration can be found at the web site having URL<www.mayoclinic.org/macular-degeneration/treatment.html>. Treatmentregimens may include chemotherapy, surgery, and/or radiation therapy.

EXAMPLES Example 1 Synthesis of Reduced Lysine Chlorotoxin Polypeptides

Reduced lysine chlorotoxin polypeptides having amino acid sequences ofSEQ ID NOs. 1-28 as shown in Table 1 and Table 2 can be synthesizedusing solid phase peptide synthesis (SPPS). Small, solid, and porousbeads are treated with linkers through which the synthesized polypeptideis covalently attached to the beads during synthesis. Nascentpolypeptides are consequently immobilized on the solid-phase andretained during washing steps.

Repeated cycles of coupling and deprotection are used to generatepolypeptides having sequences of SEQ ID NOs: 1-28. In each cycle ofcoupling and deprotection, the free N-terminal amine of a peptide orpolypeptide attached to the solid phase is coupled to a single aminoacid unit that is protected at its N-terminus by an Fmoc(9H-(f)luoren-9-yl(m)eth(o)xy(c)arbonyl) protecting group. This unit onthe growing peptide/polypeptide chain is then deprotected in basicconditions such as 20% piperidine in dimethylformamide to generate a newN-terminal amine that can be attached to a further amino acid in thenext round of coupling-deprotection.

After all cycles have been completed as desired, the polypeptide iscleaved from the bead using trifluoroacetic acid.

To generate additional reduced lysine chlorotoxin polypeptides,polypeptides having amino acid sequences of SEQ ID NOS: 1, 11, 12 and 13are modified by pegylation at lysines according to Table 2.

Example 2 Assays for Binding Activity of Reduced Lysine ChlorotoxinPolypeptides

Chlorotoxin has been shown to bind selectively to many different tumortypes including U251 glioma cells and PC3 prostate cancer cells. In thepresent Example, reduced lysine chlorotoxin polypeptides generated asdescribed in Example 2 are labeled with biotin and assayed for bindingactivity. Each biotinylated reduced lysine chlorotoxin polypeptide isincubated with U251 glioma cells and separately with PC3 prostate cancercells obtained from subconfluent cell cultures. After incubation, cellsare stained with avidin-HRP (horse-radish peroxidase) using a commercialkit according to manufacturer's instructions. Chlorotoxin is used as apositive staining control, and an incubation reaction with nopolypeptide is used as a negative staining control. A peptide with anamino acid sequence that is scrambled as compared to that of chlorotoxinmay also be used as a negative staining control. Positive staining asevidenced by presence of a colored reaction product of HRP is used as anindication of binding by the biotinylated reduced lysine chlorotoxinpolypeptide.

Reduced lysine chlorotoxin polypeptides that exhibit binding to U251 orPC3 cells are then tested (in labeled form) in competitive bindingassays in which un-labeled chlorotoxin is used as a competitor. Reducedlysine chlorotoxin polypeptides that exhibit decreasing levels ofbinding in the presence of increasing amounts of chlorotoxin areidentified.

To quantitate percentage of cells bound, avidin coupled to a fluorescentdye such as FITC or TEXAS RED™ is used instead of avidin-HRP to staincells after incubation with biotinylated polypeptides, and cells areanalyzed by FACS (fluorescence-activated cell sorting).

Example 3 Additional Assays for Binding Activity of Reduced LysineChlorotoxin Polypeptides

Reduced lysine chlorotoxin polypeptides identified as bindingcompetitively with chlorotoxin to U251 and/or PC3 in Example 2 cells arefurther assayed for binding activity to a wider range of cell types, inorder to obtain a more comprehensive binding profile.

Table 3 lists cell lines and primary cultured cells, any or anycombination of which may be tested in binding assays with reduced lysinechlorotoxin polypeptides. Cell lines and primary cultured cells listedin table 3 include both glioma and non-glioma cell lines from human,rat, and mouse.

TABLE 3 Primary Cells and Tumor Cell Lines to which binding may betested Human Glioma Cell Lines D54-MG U251-MG CH235 STTG1 U138-MG U87-MGU373-MG T98G A172 G26 Other Glioma Cell Lines C6 rat 9L rat GL2g1 mousePrimary cells Rat primary normal cortical and spinal cord astrocytecultures Human primary glioma cultures Human normal astrocyte culturesHuman normal fibroblast cultures Human umbilical vascular endothelialcells (HUVEC) Non-Glioma Cell lines SH-SY5Y human neuroblastoma SH-N-MChuman neuroblastoma HCN-2 human neuronal PFSK-1 human primitiveneuroectodermal HT 29 human colon carcinoma COS-2 monkey kidney cellline BALBc 3T3 mouse fibroblast cell line HEK 293 human epithelialkidney NIH 3T3 mouse fibroblast cell line CCD19lu human lung fibroblastH460 human lung fibroblast A549 human lung A-427 human lung carcinomaW-62 human lung cancer NIH-H1466 human lung adenocarcinoma 1299 humannon-small cell lung cell line Caco-2 human colon carcinoma HCT116 humancolon carcinoma SW948 human colorectal adenocarcinoma DU 145 humanprostate cancer PC-3 human prostate cancer LNCaP human prostate cancer2LMP human metastatic breast cancer MDA-MB-453 human breast cancerDY3672 human breast cancer HeLa human cervix carcinoma LCC6 human breastcancer HCN-2 human neuronal cell line BT474 human breast carcinomaCCD986Sk human skin fibroblast SK-BR-3 human breast adenocarcinomaMCSF-7 human breast cancer MDA-MB-231 human breast adenocarcinomaMDA-MB-468 human breast adenocarcinoma CHO Chinese hamster ovarySKMEL-31 human melanoma SKMEL-28 human melanoma Malme 3M humanmetastastic melanoma Panc-1 human pancreatic cancer PaCa-2 humanpancreatic cancer HepG2 human hepatic carcinoma Caki-1 human clear cellrenal carcinoma ACHN human renal cell adenocarcinoma Raji human lymphomaDaudi human lymphoma MOLT4 human leukemia HL60 human acute promyelocyticleukemia

Example 4 Conjugation of Monolysine Chlorotoxin Polypeptides toPaclitaxel

One or more monolysine chlorotoxin polypeptides synthesized in Example 1and optionally assayed for finding in Examples 2 and/or 3 is/areconjugated to paclitaxel, which by itself is a water insolubleanti-cancer therapeutic agent. Paclitaxel is a mitotic inhibitor.

Reduced lysine chlorotoxin polypeptides are reacted with apaclitaxel-ester-linker carboxy entity (e.g., NHS/EEDQ) in PBS andincubated. Samples from the purified final product are analyzed by highperformance liquid chromatography (HPLC) and mass spectrometry (MS) toconfirm that single species conjugates are generated. Resultingconjugates are tested for water solubility by determining the saturationconcentration and rate of solution. Conjugates that are water solubleare identified for further analysis as drug candidates.

Example 5 Conjugation of Reduced Lysine Chlorotoxin Polypeptides toGemcitabine

Reduced lysine chlorotoxin polypeptides that have no lysine residues atall (see, e.g., SEQ ID NOs: 2, 5 and 6) are synthesized as described inExample 1 and optionally assayed for binding in Examples 2 and/or 3. Thereduced lysine chlorotoxin polypeptides are conjugated to gemcitabine(GEMZAR™), a nucleoside analog, via the N-terminus.

Example 6 In Vitro Binding Assay of Chlorotoxin Conjugates

Chlorotoxin conjugates from Examples 4 and 5 can be individually testedfor binding to tumor cell lines as described for reduced lysinechlorotoxin polypeptides, as described in Examples 2 and 3.

Example 7 In Vitro Cytotoxicity Assay of Chlorotoxin Conjugates

Chlorotoxin conjugates obtained from Examples 4 and 5 and optionallytested for binding as described in Example 6 are tested for cytotoxicityin one or more cell lines listed in Table 3. Cells in culture areexposed to chlorotoxin conjugates by incubating in varyingconcentrations of chlorotoxin conjugate. After 1.5 hours of exposure,cell viability is measured and a plot of the percentage of viable cellsversus molar concentration of chlorotoxin conjugate is calculated. Forcomparison, cells are separately incubated with cytotoxic agent alone(e.g., paclitaxel from Example 4 or temozolomide from Example 5) and asimilar dose-response curve for viability is calculated.

Example 8 In Vivo Uptake of Chlorotoxin Conjugates

In vivo uptake of chlorotoxin conjugates of the present invention can beassessed by imaging using in situ radiolabeled peptide (labeled with¹³C, ²H, or ¹⁵N, etc.) and/or radiolabeled entity/moiety; nanoparticlesand magnetic resonance imaging; and/or near infrared dyes andbiophotonicimagine. Anti-chlorotoxin polypeptide antibodies may also beused to detect uptake in tissues.

Example 9 Biological Activity of Reduced Lysine Chlorotoxin Polypeptidesto Inhibit Cell Invasion

Reduced lysine chlorotoxin polypeptides (or conjugates thereof) aretested for ability to inhibit invasion of tumor cells using a Trans-wellmigration assay. In this assay, tumor cells are plated on the upperchamber of the trans-well with or without the reduced lysine chlorotoxinpolypeptide (or conjugates thereof) and migration of the cells isstimulated with a growth factor such as VEGF or PDGF. Afterapproximately 24 hours of incubation in cell culture media,non-migrating cells remaining on the upper side of the trans-well filterare removed and migrated cells on the lower side are stained forvisualization. Migrated cells are counted as an index of invasion.Modified chlorotoxin variants that have similar biological activity tochlorotoxin are considered to retain functional activity.

Example 10 Chlorotoxin Conjugates in In Vivo Breast Cancer Tumor Model

Therapeutic activity of chlorotoxin conjugates is tested in an in vivobreast cancer model. In this example, a mouse tumor model created byxenografting MDA-MB-468 breast cancer cells into recipient mice is usedto test for effects of paclitaxel-chlorotoxin conjugates generated inExamples 4 on tumor growth.

Mice bearing flank tumors are injected intravenously at a paclitaxeldose of 3.7 mg/kg three times per week for a total of 8 doses. Theconcentration of conjugate being used in these experiments is“sub-therapeutic” in that the same dosing regimen using non-conjugatedpaclitaxel is not enough to have a therapeutic effect. A group of miceis injected with saline alone as a control. Another group of mice isinjected with paclitaxel at a dose of 3.7 mg/kg. Tumor growth ismonitored using calipers to measure the length and width of the tumorduring and after the treatment interval. Tumor growth over time isplotted as percent of original tumor size versus time.

OTHER EMBODIMENTS

Other embodiments of the invention will be apparent to those skilled inthe art from a consideration of the specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope of theinvention being indicated by the following claims.

The invention claimed is:
 1. A chlorotoxin polypeptide, wherein thechlorotoxin polypeptide has at least 90% sequence identity with SEQ IDNO: 1, and wherein each lysine residue present in the chlorotoxinpolypeptide, except for the lysine residue corresponding to position 27of SEQ ID NO:1, is blocked from participating in a chemical conjugationreaction by a modification to the lysine residue.
 2. The chlorotoxinpolypeptide of claim 1, wherein the modification is a covalentmodification or a non-covalent modification.
 3. The chlorotoxinpolypeptide of claim 2, wherein the covalent modification is pegylationor methylation.
 4. The chlorotoxin polypeptide of claim 1, wherein atleast one blocked lysine residue is modified at the epsilon NH₂ group ofthe lysine residue.
 5. A conjugate comprising the chlorotoxinpolypeptide of claim 1 associated with at least one therapeutic agent,at least one detection agent, or combination thereof.
 6. The conjugateof claim 5, wherein the chlorotoxin polypeptide and the therapeuticagent or the detection agent are directly covalently associated.
 7. Theconjugate of claim 5, wherein the chlorotoxin polypeptide and thetherapeutic agent or the detection agent are covalently associatedthrough a linker.
 8. The conjugate of claim 5, wherein the therapeuticagent is an anti-cancer agent.
 9. The conjugate of claim 5, wherein thetherapeutic agent is a member of the group consisting of radioisotopes,prodrug activating enzymes, radiosensitizers, interfering ribonucleicacids (“RNAs”), superantigens, anti-angiogenic agents, alkylatingagents, purine antagonists, pyrimidine antagonists, plant alkaloids,intercalating antibiotics, aromatase inhibitors, anti-metabolites,mitotic inhibitors, growth factor inhibitors, cell cycle inhibitors,enzymes, topoisomerase inhibitors, biological response modifiers,anti-hormones and anti-androgens.
 10. The conjugate of claim 5, whereinthe detection agent is a fluorescent label, a radiolabel, a magneticresonance imaging label, or a combination thereof.
 11. A pharmaceuticalcomposition comprising the conjugate of claim 5 and a pharmaceuticallyacceptable carrier.